WO2023078021A1 - Anticorps monoclonal anti-bcma et conjugué anticorps-médicament - Google Patents

Anticorps monoclonal anti-bcma et conjugué anticorps-médicament Download PDF

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WO2023078021A1
WO2023078021A1 PCT/CN2022/123901 CN2022123901W WO2023078021A1 WO 2023078021 A1 WO2023078021 A1 WO 2023078021A1 CN 2022123901 W CN2022123901 W CN 2022123901W WO 2023078021 A1 WO2023078021 A1 WO 2023078021A1
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antibody
och
independently
alkyl
derivatives
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PCT/CN2022/123901
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English (en)
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Robert Yongxin Zhao
Junxiang JIA
Yunxia ZHENG
Qingliang YANG
Yuanyuan Huang
Lingli Zhang
Wenjun Li
Huihui GUO
Hangbo YE
Zhicang YE
You Zhou
Juan Wang
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Hangzhou Dac Biotech Co., Ltd.
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Priority to AU2022383265A priority Critical patent/AU2022383265A1/en
Priority to CA3236852A priority patent/CA3236852A1/fr
Priority to PCT/CN2022/129122 priority patent/WO2023078273A1/fr
Priority to CA3236754A priority patent/CA3236754A1/fr
Priority to AU2022381163A priority patent/AU2022381163A1/en
Priority to CN202280069631.1A priority patent/CN118215676A/zh
Publication of WO2023078021A1 publication Critical patent/WO2023078021A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6867—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of a blood cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives

Definitions

  • This application includes an electronic sequence listing in a file named FE00688PCT-Sequence listing. xml created on October 10, 2022 and containing 40 KB, which is hereby incorporated by reference.
  • BCMA The B cell maturation antigen
  • TNF tumor necrosis factor
  • BCMA binds to two distinct ligands, B cell activating factor (BAFF; also known as BlyS, TALL-1, TNFSF13B, and THANK) and a proliferation-inducing ligand (APRIL, TNFSF13) (Schiemann, B, et al, Science. 2001, 293 (5537) : 2111-4; Vidal-Laliena, M. et al, Cell Immunol.
  • BAFF B cell activating factor
  • APRIL proliferation-inducing ligand
  • BCMA transmembrane activator and calcium modulator and cyclophilin ligand interactor
  • BAFF-R BAFF receptor
  • BCMA has been linked to a number of cancers, autoimmune disorders, and infectious diseases (Coquery, C.M. and Erickson, L.D. Crit. Rev. Immunol. 2012; 32 (4) : 287-305) .
  • BCMAprotein is highly expressed on the surface of plasma cells from multiple myeloma patients (Novak et al, Blood, 103 (2) : 689-694 (2004) ; Neri et al., Clinical Cancer Research, 73 (19) : 5903-5909 (2007) ; and Moreaux et al., Blood, 703 (8) : 3148-3157 (2004) ) .
  • BCMA wasextensively investigated as a therapeutic target for multiple myeloma and autoimmune disorders during the past decade (Ni, B. and Hou, J., Hematology. 2022, 27 (1) : 343-352; Tan, C.R. and Shah, U.A., CurrHematolMalig Rep. 2021, 16 (5) : 367-383) .
  • MM Multiple Myeloma
  • induction/neoadjuvant therapy including chemotherapy, radiation, surgery, biophosphonates
  • PI proteasome inhibitor
  • IMID immunomodulator
  • ASCT autologous (hematopoietic) stem cell transplantation
  • MM myeloma
  • BCMAantibody and an antibody-drug conjugate comprising a BCMA monoclonal antibody, or a BCMA antigen-binding fragment thereof, conjugated to a cytotoxin, directed against B-cell maturation antigen (BCMA) .
  • the BCMA antibodies and its ADCV are useful for treatment and diagnoses of various cancers, autoimmune disorders, and infectious diseases as well as detecting BCMA.
  • This invention also continues to applythe methodology of specific conjugation (PCT/CN2021/128453) to construct these BCMA ADCs. Further disclosed are pharmaceutical compositions, screening and medical treatment methods.
  • the present invention provides antigen binding proteins which specifically bind to BCMA (CD269) , for example antibodies which specifically bind to BCMA and which inhibit the binding of BAFF and/or APRIL to the BCMA receptor.
  • the present invention also provides antigen binding proteins which specifically bind to BCMA and which inhibits the binding of BAFF and/or APRIL to BCMA and wherein the antigen binding protein is capable of internalization.
  • the BCMA monoclonal antibody comprises (a) a heavy chain variable region comprising a complementarity determining region 1 (HCDR1) amino acid sequence of SEQ ID NO: 1, an HCDR2 amino acid sequence of SEQ ID NO: 2, and an HCDR3 amino acid sequence of SEQ ID NO: 3 and (b) a light chain variable region comprising a complementarity determining region 1 (LCDR1) amino acid sequence of SEQ ID NO: 4, an LCDR2 amino acid sequence of SEQ ID NO: 5, and an LCDR3 amino acid sequence of SEQ ID NO: 6.
  • the present invention also provides an antibody-drug conjugate (ADC) comprising a monoclonal antibody, or an antigen-binding fragment thereof, directed against B-cell maturation antigen (BCMA) conjugated to a cytotoxin.
  • ADC antibody-drug conjugate
  • BCMA B-cell maturation antigen
  • the antigen binding proteins are conjugated to a toxin such as atubulysin analog, a PBD dimer or an auristatin analog.
  • compositions comprising the foregoing antibody-drug conjugate, and a pharmaceutically acceptable carrier, and methods of killing multiple myeloma cells (including multiple myeloma stem cells) that express BCMA by contacting multiple myeloma cells with the ADC.
  • a method of treating a human patient afflicted with a B cell related disorders or diseases such as antibody mediated or plasma cell mediated diseases or plasma cell malignancies such as for example Multiple Myeloma (MM) which method comprises the step of administering to said patient a therapeutically effective amount of the antigen binding antibody and/or ADC thereof as described herein.
  • a B cell related disorders or diseases such as antibody mediated or plasma cell mediated diseases or plasma cell malignancies such as for example Multiple Myeloma (MM)
  • MM Multiple Myeloma
  • a method of treating a human patient afflicted with Rheumatoid Arthritis, Psoriasis, Type 1 Diabetes Mellitus or Multiple Sclerosis comprises the step of administering to said patient a therapeutically effective amount of the antigen binding protein and/or ADCas described herein.
  • Fig. 1 shows binding affinity of hybridoma antibody BCMA-A2-6H4-5D2 and positive control antibody J6M0 to recombinant expressed TrxA-BCMA.
  • Fig. 2 shows Binding affinity of hybridoma antibody BCMA-A2-6H4-5D2, chimeric antibody c5D2, humanized antibody hu5D2 and positive control antibody J6M0 to recombinant expressed TrxA-BCMA.
  • Fig. 3 shows Binding affinity of humanized antibody hu5D2, hu5D2 conjugated ADC and isotype control antibody to endogenous BCMA expressed cell line NCI-H929.
  • Fig. 4A Illustrates the killing of BCMA over-expressed RPMI-8226 cell lines by the antibody drug conjugate, hu5D2-tubulysin B analog conjugate (C-390) .
  • Fig. 4 Illustrates the killing of cell line NCI-H929 by the antibody drug conjugate: hu5D2-tubulysin B analog conjugate (C-390) , in comparison to the ADC J6M0-tubulysin B analog conjugate (C-390) , naked hu5D2 antibody, unconjugated tubulysin B analog (compound 390) and Paclitaxel.
  • Fig. 4DIl lustrates the killing of BCMA negative expression cell line Jurkatby the antibody drug conjugate hu5D2-tubulysin B analog conjugate (C-390) , in comparisonwith the ADC J6M0-tubulysin B analog conjugate (C-390) , naked hu5D2 antibody, unconjugated tubulysin B analog (compound 390) and Paclitaxel.
  • Fig. 4EIl lustrates the killing of BCMA expression cell line U266B1 by the BCMA antibody (hu5D2) -drug conjugates: C-221, C-202, C-88, C-326, C-30, in comparisonwith Paclitaxel.
  • Fig. 5 Illustrate MS/MS daughter or product ion spectrum of glycopeptides of the BCMA antibody.
  • (a) Non-glycosylated peptides;
  • (b) Man5 containing glycopeptides;
  • (c) G0F-GlcNAc containing glycopeptides;
  • (d) G0 containing glycopeptides;
  • (e) G0F containing glycopeptides;
  • g) G1F containing glycopeptides;
  • (h) G2F containing glycopeptides.
  • Light chains (LC) with zero or one drug molecule attached (L0 and L1)
  • (b) heavy chains with zero, one, two, or three drug molecules attached H0, H1, H2 and H3 .
  • BCMA hu5D2
  • ADCs C-68a, C-115, C-192, C-202, C-221, C-290, C-306, C-385, C-390, C-399, C-402, C-417, DARs indicated in table 7
  • BCMA-mcMMAF belantamabmcMMAF
  • PBS buffer the control
  • Fig. 10 Illustrates change in tumor volume in a JJN-3cell xenograft mouse model of multiple myeloma in response to a serial of single dose (5 mg/Kg) treatment with hu5D2-ADC in comparison to belantamabmcMMAFand PBS buffer (the control) .
  • the figure indicates that all the 9 conjugates had antitumor activity, and the orders of the antitumor activity are: Paclitaxel ⁇ C-385 ⁇ belantamabmcMMAF ⁇ C-195 ⁇ C-137 ⁇ C-181b ⁇ C-126 ⁇ C-83 ⁇ C-277 ⁇ C-258.
  • Fig. 11 Illustrates change in tumor volume in NCI-H929 cell xenograft mouse model of multiple myeloma in response to a serial of single dose (2 mg/Kg) treatment with hu5D2-ADC in comparison to belantamabmcMMAF and PBS buffer (the control) .
  • the figure indicates that all the 7 conjugates had antitumor activity, and the orders of the antitumor activity are: belantamabmcMMAF ⁇ C-406 ⁇ C-396 ⁇ C-399 ⁇ C-400 ⁇ C-221b ⁇ C-402.
  • Fig. 12 shows the general synthesis ofcomponents of a bis-linker.
  • Fig. 13 shows the synthesis of a camptothecin analogcontaining a bis-conjugate linker.
  • Fig. 14 shows the synthesis of acamptothecin analogcontaining a bis-conjugate linker.
  • Fig. 15 shows the synthesis of a camptothecin analogcontaining a bis-conjugate linker.
  • Fig. 16 shows the general synthesis ofcomponents of a bis-linker.
  • Fig. 17 shows the synthesis of a camptothecin analogcontaining a bis-conjugate linker.
  • Fig. 18 shows the synthesis of a camptothecin analogcontaining a bis-conjugate linker.
  • Fig. 19 shows the synthesis of a camptothecin analogcontaining a bis-conjugate linker.
  • Fig. 20 shows the general synthesis ofcomponents of a bis-linker.
  • Fig. 21 shows the synthesis of a camptothecin analogcontaining a bis-conjugate linker.
  • Fig. 22 shows the synthesis of atubulysin B analogcontaining a bis-conjugate linker.
  • Fig. 23 shows the synthesis of a tubulysin B analogcontaining a bis-conjugate linker.
  • Fig. 24 shows the synthesis of a tubulysin B analogcontaining a bis-conjugate linker.
  • Fig. 25 shows the synthesis of a tubulysin B analogcontaining a bis-conjugate linker.
  • Fig. 26 shows the synthesis of a tubulysin B analogcontaining a bis-conjugate linker.
  • Fig. 27 shows the synthesis of a tubulysin B analogcontaining a bis-conjugate linker.
  • Fig. 28 shows the synthesis ofcomponents of a tubulysin B analogs.
  • Fig. 29 shows the synthesis of a tubulysin B analogcontaining a bis-conjugate linker.
  • Fig. 30 shows the synthesis of a tubulysin B analogcontaining a bis-conjugate linker.
  • Fig. 31 shows the synthesis of a camptothecin analogcontaining a bis-conjugate linker.
  • Fig. 32 shows the synthesis of a camptothecin analogcontaining a bis-conjugate linker.
  • Fig. 33 shows the synthesis of a camptothecin analogcontaining a bis-conjugate linker.
  • Fig. 34 shows the synthesis of a camptothecin analogcontaining a bis-conjugate linker.
  • Fig. 35 shows the synthesis of a camptothecin analogcontaining a bis-conjugate linker.
  • Fig. 36 shows the synthesis ofa camptothecin analogcontaining a bis-conjugate linker and components of amanitin analogs.
  • Fig. 37 shows the synthesis ofan amanitin analogcontaining a bis-conjugate linker.
  • Fig. 38 shows the synthesis ofan amanitin analogcontaining a bis-conjugate linker.
  • Fig. 39 shows the synthesis ofan amanitin analogcontaining a bis-conjugate linker.
  • Fig. 40 shows the synthesis ofan amanitin analogcontaining a bis-conjugate linker.
  • Fig. 41 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Fig. 42 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Fig. 43 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Fig. 44 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Fig. 45 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Fig. 46 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Fig. 47 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Fig. 48 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Fig. 49 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Fig. 50 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Fig. 51 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Fig. 52 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Fig. 53 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Fig. 54 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Fig. 55 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Fig. 56 shows the synthesis of a tubulysin B analogcontaining a linker and components of PBD analogs.
  • Fig. 57 shows the synthesis of a PBD analogcontaining a linker.
  • Fig. 58 shows the synthesis of a PBD analogcontaining a linker.
  • Fig. 59 shows the synthesis of a PBD analogcontaining a linker and a tubulysin B analogcontaining a linker.
  • Fig. 60 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Fig. 61 shows the synthesis of a tubulysin B analogcontaining a linker.
  • Alkyl refers to an aliphatic hydrocarbon group or univalent groups derived from alkane by removal of one or two hydrogen atoms from carbon atoms. It may be straight or branched having C 1 -C 8 (1 to 8 carbon atoms) in the chain. “Branched” means that one or more lower C numbers of alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain.
  • Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2, 2-dimethylbutyl, 2, 3-dimethylbutyl, 2, 2-dimethylpentyl, 2, 3-dimethylpentyl, 3, 3-dimethylpentyl, 2, 3, 4-trimethylpentyl, 3-methyl-hexyl, 2, 2-dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 3, 5-dimethylhexyl, 2, 4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, and isooctyl.
  • a C 1 -C 8 alkyl group can be unsubstituted or substituted with one or more groups including, but not limited to, -C 1 -C 8 alkyl, -O- (C 1 -C 8 alkyl) , -aryl, -C (O) R', -OC (O) R', -C (O) OR', -C (O) NH 2 , -C (O) NHR', -C (O) N (R') 2 , -NHC (O) R', -SR', -S (O) 2 R', -S (O) R', -OH, -halogen, -N 3 , -NH 2 , -NH (R') , -N (R') 2 and -CN; where each R' is independently selected from -C 1 -C 8 alkyl and aryl.
  • Halogen refers to fluorine, chlorine, bromine or iodine atom; preferably fluorine and chlorine atom.
  • Heteroalkyl refers to C 2 -C 8 alkyl in which one to four carbon atoms are independently replaced with a heteroatom from the group consisting of O, S and N.
  • Carbocycle refers to a saturated or unsaturated ring having 3 to 8 carbon atoms as a monocycle or 7 to 13 carbon atoms as a bicycle.
  • Monocyclic carbocycles have 3 to 6 ring atoms, more typically 5 or 6 ring atoms.
  • Bicyclic carbocycles have 7 to 12 ring atoms, arranged as a bicycle [4, 5] , [5, 5] , [5, 6] or [6, 6] system, or 9 or 10 ring atoms arranged as a bicycle [5, 6] or [6, 6] system.
  • Representative C 3 -C 8 carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1, 3-cyclohexadienyl, -1, 4-cyclohexadienyl, -cycloheptyl, -1, 3-cycloheptadienyl, -1, 3, 5-cycloheptatrienyl, -cyclooctyl, and -cyclooctadienyl.
  • a “C 3 -C 8 carbocycle” refers to a 3-, 4-, 5-, 6-, 7-or 8-membered saturated or unsaturated nonaromatic carbocyclic ring.
  • a C 3 -C 8 carbocycle group can be unsubstituted or substituted with one or more groups including, but not limited to, -C 1 -C 8 alkyl, -O- (C 1 -C 8 alkyl) , -aryl, -C (O) R', -OC (O) R', -C (O) OR', -C (O) NH 2 , -C (O) NHR', -C (O) N (R') 2 , -NHC (O) R', -SR', -S (O) R', -S (O) 2 R', -OH, -halogen, -N 3 , -NH 2 , -NH (R') , -N (R') 2 and
  • Alkenyl refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond which may be straight or branched having 2 to 8 carbon atoms in the chain.
  • alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, hexylenyl, heptenyl, octenyl.
  • Alkynyl refers to an aliphatic hydrocarbon group containing a carbon-carbon triple bond which may be straight or branched having 2 to 8 carbon atoms in the chain.
  • exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, n-pentynyl, hexylynyl, heptynyl, and octynyl.
  • Alkylene refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane.
  • Typical alkylene radicals include, but are not limited to: methylene (-CH 2 -) , 1, 2-ethyl (-CH 2 CH 2 -) , 1, 3-propyl (-CH 2 CH 2 CH 2 -) , 1, 4-butyl (-CH 2 CH 2 CH 2 CH 2 -) , and the like.
  • Alkenylene refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkene.
  • Alkynylene refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkyne.
  • Typical alkynylene radicals include, but are not limited to: acetylene, propargyl and 4-pentynyl.
  • Aryl or Ar refers to an aromatic or hetero aromatic group, composed of one or several rings, comprising three to fourteen carbon atoms, preferentially six to ten carbon atoms.
  • hetero aromatic group refers one or several carbon on aromatic group, preferentially one, two, three or four carbon atoms are replaced by O, N, Si, Se, P or S, preferentially by O, S, and N.
  • Heterocycle refers to a ring system in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group of O, N, S, Se, B, Si and P. Preferable heteroatoms are O, N and S. Heterocycles are also described in The Handbook of Chemistry and Physics, 78th Edition, CRC Press, Inc., 1997-1998, p. 225 to 226, the disclosure of which is hereby incorporated by reference.
  • Preferred nonaromatic heterocyclic include epoxy, aziridinyl, thiiranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxolanyl, piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl, as well as the fused
  • heteroaryl refers to a 3 to 14, preferably 5 to 10 membered aromatic hetero, mono-, bi-, or multi-cyclic ring.
  • examples include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1, 2, 4-thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, pyridyl-N-oxide, as well as the fused systems resulting from the condensation with a phenyl group
  • Alkyl “, “cycloalkyl “, “alkenyl “, “alkynyl “, “aryl “, “heteroaryl “, “heterocyclic” and the like refer also to the corresponding “alkylene “, “cycloalkylene “, “alkenylene “, “alkynylene “, “arylene “, “heteroarylene “, “heterocyclene” and the likes which are formed by the removal of two hydrogen atoms.
  • Arylalkyl refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with an aryl radical.
  • Typical arylalkyl groups include, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and the like.
  • Heteroarylalkyl refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with a heteroaryl radical.
  • heteroarylalkyl groups are 2-benzimidazolylmethyl, 2-furylethyl.
  • Examples of a “hydroxyl protecting group” includes, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, t-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetate ester, substituted acetate esters, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.
  • leaving group refers to a functional group that can be substituted by another functional group.
  • Such leaving groups are well known in the art, and examples include, a halide (e.g., chloride, bromide, and iodide) , methanesulfonyl (mesyl) , p-toluenesulfonyl (tosyl) , trifluoro-methylsulfonyl (triflate) , and trifluoromethylsulfonate.
  • a preferred leaving group is selected from nitrophenol; N-hydroxysuccinimide (NHS) ; phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3′-sulfonate, anhydrides formed its self, or formed with the other anhydride, e.g. acetyl anhydride, formyl anhydride; or an intermediate molecule generated with a condensation reagent for peptide coupling reactions or for Mitsunobu reactions.
  • NHS N-hydroxysuccinimide
  • Boc tert-butoxy carbonyl
  • BroP bromotrispyrrolidinophosphonium hexafluorophosphate
  • CDI 1, 1'-carbonyldiimidazole
  • DCC dicyclohexylcarbodiimide
  • DCE dichloroethane
  • DCM dichloromethane
  • DIAD diisopropylazodicarboxylate
  • DIBAL-H diisobutyl-aluminium hydride
  • DIPEA diisopropylethylamine
  • DEPC diethyl phosphorocyanidate
  • DMA N, N-dimethyl acetamide
  • DMAP 4- (N, N-dimethylamino) pyridine
  • DMF N, N-dimethylformamide
  • DMSO dimethylsulfoxide
  • DTT dithiothreitol
  • EDC 1- (3-dimethylamino)
  • amino acid (s) can be natural and/or unnatural amino acids, preferably alpha-amino acids.
  • Natural amino acids are those encoded by the genetic code, which are alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine. tryptophan and valine.
  • the unnatural amino acids are derived forms of proteinogenic amino acids.
  • Examples include hydroxyproline, lanthionine, 2-aminoisobutyric acid, dehydroalanine, gamma-aminobutyric acid (the neurotransmitter) , ornithine, citrulline, beta alanine (3-aminopropanoic acid) , gamma-carboxyglutamate, selenocysteine (present in many noneukaryotes as well as most eukaryotes, but not coded directly by DNA) , pyrrolysine (found only in some archaea and one bacterium) , N-formylmethionine (which is often the initial amino acid of proteins in bacteria, mitochondria, and chloroplasts) , 5-hydroxytryptophan, L-dihydroxyphenylalanine, triiodothyronine, L-3, 4-dihydroxyphenylalanine (DOPA) , and O-phosphoserine.
  • DOPA 4-dihydroxyphenylalanine
  • amino acid also includes amino acid analogs and mimetics.
  • Analogs are compounds having the same general H 2 N (R) CHCO 2 H structure of a natural amino acid, except that the R group is not one found among the natural amino acids. Examples of analogs include homoserine, norleucine, methionine-sulfoxide, and methionine methyl sulfonium.
  • an amino acid mimetic is a compound that has a structure different from the general chemical structure of an alpha-amino acid but functions in a manner similar to one.
  • the term “unnatural amino acid” is intended to represent the “D” stereochemical form, the natural amino acids being of the “L” form.
  • amino acid sequence is then preferably a cleavage recognition sequence for a protease.
  • Many cleavage recognition sequences are known in the art. See, e.g., Matayoshi et al. Science 247: 954 (1990) ; Dunn et al. Meth. Enzymol. 241: 254 (1994) ; Seidah et al. Meth. Enzymol. 244: 175 (1994) ; Thornberry, Meth. Enzymol. 244: 615 (1994) ; Weber et al. Meth. Enzymol. 244: 595 (1994) ; Smith et al. Meth. Enzymol.
  • sequence is selected from the group consisting of Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Lys, Cit, Ser, and Glu.
  • “Pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
  • “Pharmaceutically acceptable solvate” or “solvate” refer to an association of one or more solvent molecules and a disclosed compound.
  • solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid and ethanolamine.
  • “Pharmaceutically acceptable excipient” includes any carriers, diluents, adjuvants, or vehicles, such as preserving or antioxidant agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • preserving or antioxidant agents such as preserving or antioxidant agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions as suitable therapeutic combinations.
  • pharmaceutically acceptable salts refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof.
  • the pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, tartaric, citric, methanesulfonic, benzenesulfonic, glucuronic, glutamic, benzoic, salicylic, toluenesulfonic, oxalic, fumaric, maleic, lactic and the like.
  • Further addition salts include ammonium salts such as tromethamine, meglumine, epolamine, etc., metal salts such as sodium, potassium, calcium, zinc or magnesium.
  • the pharmaceutical salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
  • such salts can be prepared via reaction the free acidic or basic forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.
  • non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17 th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418, the disclosure of which is hereby incorporated by reference.
  • administering refers to any mode of transferring, delivering, introducing or transporting a pharmaceutical drug or other agent to a subject. Such modes include oral administration, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous or intrathecal administration. Also contemplated by the present invention is utilization of a device or instrument in administering an agent. Such device may utilize active or passive transport and may be slow-release or fast-release delivery device.
  • ACES N- (2-Acetamido) -2-aminoethanesulfonic acid
  • ADA N- (2-Acetamido) iminodiacetic acid, N- (Carbamoylmethyl) iminodiacetic acid
  • pH 6.0-7.2 pH 6.0-7.2
  • pKa 6.65
  • AMPD (2-amino-2-methyl-1, 3-propanediol) ) is a useful buffer at pH 7.8 -9.7.
  • Bicine N, N-Bis (2-hydroxyethyl) glycine
  • BisTris propane (1, 3-Bis [tris (hydroxymethyl) methylamino] propane) .
  • DIPSO N, N-Bis (2-hydroxyethyl) -3-amino-2-hydroxypropanesulfonic acid
  • HEBPS N- (2-Hydroxyethyl) piperazine-N′- (4-butanesulfonic acid)
  • pKa 8.30
  • HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid ; 2-morpholinoethanesulfonic acid; 2- (4-morpholino) ethanesulphonic acid; 2- (N-morpholino) ethanesulfonic acid; morpholine-4-ethanesulfonic acid hydrate) is widely used to buffer at pH 6.8 -8.2; pKa at 20°C: 7.45-7.65)
  • HEPPSO (2-Hydroxyethyl) piperazine-1- (2-hydroxypropanesulfonic acid) hydrate
  • MES (2- (N-morpholino) ethanesulfonic acid, monohydrate) is used as buffering agent at pH 5.2-7.1 (pKa: 6.16) .
  • MOBS (4-Morpholinebutanesulfonic acid; 3- (N-Morpholino) butanesulfonic acid hemisodium salt) is an homolog of MES and MOPS with higher pKa/It is used to buffer solution at pH6.9-8.3 (pKa: 7.6) .
  • MOPS (4-Morpholinepropanesulfonic acid sodium salt) .
  • MOPSO ⁇ -Hydroxy-4-morpholinepropanesulfonic acid, 3-Morpholino-2-hydroxypropanesulfonic acid
  • POPSO Piperazine-1, 4-bis (2-hydroxypropanesulfonic acid) dihydrate
  • TAPS [ (2-Hydroxy-1, 1-bis (hydroxymethyl) ethyl) amino] -1-propanesulfonic acid
  • TAPSO (2-Hydroxy-3- [tris (hydroxymethyl) methylamino] -1-propanesulfonic acid) .
  • Tricine (Piperazine-N, N'-Bis [2-Hydroxypropanesulfonic Acid) ] is used to buffer at pH7.4-8.8 (pKa: 8.16) .
  • antibody is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) , and antibody fragments so long as they exhibit the desired antigen-binding activity and fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site.
  • An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof) , and the antibody need not be of any particular class.
  • immunoglobulins can be assigned to different classes.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes) , e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F (ab') 2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv) ; and multispecific antibodies formed from antibody fragments.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
  • the term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) .
  • FRs conserved framework regions
  • HVRs hypervariable regions
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993) ; Clarkson et al., Nature 352: 624-628 (1991) .
  • “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes) , each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256: 495, 1975, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567.
  • the monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., Nature 348: 552-554, 1990, for example.
  • humanized antibody refers to forms of non-human (e.g. murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F (ab') 2 or other antigen binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementarity determining region
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc) , typically that of a human immunoglobulin.
  • CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3 are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody.
  • human antibody means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or which has been made using any of the techniques for making human antibodies known to those skilled in the art or disclosed herein.
  • This definition of a human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide.
  • One such example is an antibody comprising murine light chain and human heavy chain polypeptides.
  • Human antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et al., Nature Biotechnology, 14: 309-314, 1996; Sheets et al., Proc. Natl. Acad.
  • Human antibodies can also be made by immunization of animals into which human immunoglobulin loci have been transgenically introduced in place of the endogenous loci, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.
  • the human antibody may be prepared by immortalizing human B lymphocytes that produce an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or from single cell cloning of the cDNA, or may have been immunized in vitro) . See, e.g., Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol., 147 (1) : 86-95, 1991; and U.S. Pat. No. 5,750,373.
  • chimeric antibody is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
  • polypeptide oligopeptide
  • peptide peptide and protein are used interchangeably herein to refer to chains of amino acids of any length, preferably, relatively short (e.g., 10-100 amino acids) .
  • the chain may be linear or branched, it may comprise modified amino acids, and/or may be interrupted by non-amino acids.
  • the terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • polypeptides can occur as single chains or associated chains.
  • a “monovalent antibody” comprises one antigen binding site per molecule (e.g., IgG or Fab) .
  • a monovalent antibody can have more than one antigen binding sites, but the binding sites are from different antigens.
  • a “monospecific antibody” comprises two identical antigen binding sites per molecule (e.g. IgG) such that the two binding sites bind identical epitope on the antigen. Thus, they compete with each other on binding to one antigen molecule. Most antibodies found in nature are monospecific. In some instances, a monospecific antibody can also be a monovalent antibody (e.g. Fab) .
  • bivalent antibody comprises two antigen binding sites per molecule (e.g., IgG) . In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies may be bispecific.
  • bispecific or dual-specific is a hybrid antibody having two different antigen binding sites.
  • the two antigen binding sites of a bispecific antibody bind to two different epitopes, which may reside on the same or different protein targets.
  • a “bifunctional” is antibody is an antibody having identical antigen binding sites (i.e., identical amino acid sequences) in the two arms but each binding site can recognize two different antigens.
  • heteromultimer is a molecule comprising at least a first polypeptide and a second polypeptide, wherein the second polypeptide differs in amino acid sequence from the first polypeptide by at least one amino acid residue.
  • the heteromultimer can comprise a “heterodimer” formed by the first and second polypeptide or can form higher order tertiary structures where polypeptides in addition to the first and second polypeptide are present.
  • heterodimer is a molecule comprising a first polypeptide and a second polypeptide, wherein the second polypeptide differs in amino acid sequence from the first polypeptide by at least one amino acid residue.
  • the “hinge region” includes the meaning known in the art, which is illustrated in, for example, Janeway et al., ImmunoBiology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999) ; Bloom et al., Protein Science (1997) , 6: 407-415; Humphreys et al., J. Immunol. Methods (1997) , 209: 193-202.
  • immunoglobulin-like hinge region refers to the hinge region and hinge sequence of an immunoglobulin-like or an antibody-like molecule (e.g., immunoadhesins) .
  • the immunoglobulin-like hinge region can be from or derived from any IgG1, IgG2, IgG3, or IgG4 subtype, or from IgA, IgE, IgD or IgM, including chimeric forms thereof, e.g., a chimeric IgG1/2 hinge region.
  • immune effector cell refers to a cell within the natural repertoire of cells in the human immune system which can be activated to affect the viability of a target cell.
  • the viability of a target cell can include cell survival, proliferation, and/or ability to interact with other cells.
  • Antibodies of the invention can be produced using techniques well known in the art, e.g., recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art (see, for example, Jayasena, S.D., Clin. Chem., 45: 1628-50, 1999 and Fellouse, F.A., et al, J. Mol. Biol., 373 (4) : 924-40, 2007) .
  • cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, I131, I125, Y90, In111, Re186, Re188, Sm153, Bi212, P32, Pb212, Zr89, F18, and radioactive isotopes of Lu, e.g.
  • chemotherapeutic agents or drugs e.g., tubulysin, maytansin, auristatin, DNA minor groove binders (such as PBD dimers) , ducarmysin, topoisomerase inhibitor, RNA polymerase inhibitors, DNA alkylators, methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide) , doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents) ; growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed throughout the application.
  • Linker refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody to a drug moiety.
  • linkers include a divalent radical such as an alkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as: -- (CR 2 ) nO (CR 2 ) n--, repeating units of alkyloxy (e.g. polyethylenoxy, PEG, polymethyleneoxy) and alkylamino (e.g. polyethyleneamino) ; and diacid ester and amides including succinate, succinamide, diglycolate, malonate, and caproamide.
  • linkers can comprise one or more amino acid residues, such as valine, phenylalanine, lysine, and homolysine.
  • BCMA means a human BCMA.
  • Exemplary human nucleic acid and amino acid sequences are provided by SEQ ID Nos: 1 and 2.
  • BMCA means at least an extracellular domain of the protein (approximately residues 1-54 of SEQ ID NO: 7) and sometimes the complete protein.
  • the present invention provides a method for the treatment of a medical disorder in a human subject, wherein the medical disorder is associated with the presence of pathogenic B cells expressing B cell maturation antigen (BCMA) , the method comprising administering to the human subject an isolated monoclonal antibody or an antigen binding fragment thereof that binds BCMA (CD269) .
  • BCMA B cell maturation antigen
  • the present BCMA antibody (e. q. hu5D2) is a humanized monoclonal antibody that specifically binds to human BCMA as described in the examples.
  • the 5D2 antibody was producedbyhybridomaBCMA-A2-6H4-5D2.
  • a deposit at China Center for Type Culture Collection (CCTCC) was made on June23, 2022 under the Budapest Treaty.
  • the CCTCC is located at Wuhan University, Wuhan City, Hubei, Post code 430000, P. R. China.
  • the CCTCC deposit was assigned accession number of CCTCC C2022188.
  • Hu5D2antibody inhibits binding of BCMA to both of its ligands, APRIL and BAFF.
  • the Hu5D2antibody can also be incorporated into an antibody drug conjugate to deliver a linked drug into the interior of cells expressing BCMA.
  • the Hu5D2antibody is another humanized monoclonal antibody that specifically binds to human BCMA, inhibits its binding to its ligands and can deliver a linked drug to the interior of cells expressing BCMA.
  • the present invention provides antigen binding proteins which bind to membrane bound targets and wherein the antigen binding protein is capable of internalisation.
  • an immunoconjugate comprising the antigen binding protein of the present invention and a cytotoxic agent.
  • the antigen binding protein has ADCC effector function for example the antigen binding protein has enhanced ADCC effector function.
  • antigen binding proteins or fragments thereof which specifically bind to BCMA, for example which specifically binds human BCMA (hBCMA) and which inhibit the binding of BAFF and/or APRIL to the BCMA receptor.
  • hBCMA human BCMA
  • the antigen binding proteins or fragments of the present invention specifically bind to BCMA and inhibit the binding of BAFF and/or APRIL to BCMA wherein the antigen binding proteins or fragments thereof have the ability to bind to Fc ⁇ RIIIA and mediate FcgRIIIA mediated effector functions, or have enhanced Fc. ⁇ RIIIA mediated effector function.
  • the antigen binding proteins are capable of internalisation.
  • an antigen binding protein according to the invention as herein described which binds to non-membrane bound BCMA, for example to serum BCMA.
  • an antigen binding protein as herein described wherein the antigen binding protein comprises CDRH3 of SEQ ID NO. 3 or a variant of SEQ ID NO. 3.
  • an antigen binding protein as herein described wherein the antigen binding protein further comprises one or more of: CDR H1 of SEQ. ID. NO:1, CDRH2: SEQ. ID. NO: 2: CDRL1: SEQ. ID. NO: 4, CDRL2: SEQ. ID. NO: 5 and/or CDRL3: SEQ. ID. NO: 6 and or variants thereof.
  • the antigen binding proteins of the present invention may comprise heavy chain variable regions and light chain variable regions of the invention which may be formatted into the structure of a natural antibody or functional fragment or equivalent thereof.
  • An antigen binding protein of the invention may therefore comprise the VH regions of the invention formatted into a full length antibody, a (Fab') 2 fragment, a Fab fragment, or equivalent thereof (such as scFV, bi-tri-or tetra-bodies, Tandabs etc. ) , when paired with an appropriate light chain.
  • the antibody may be an IgG1, IgG2, IgG3, or IgG4; or IgM; IgA, IgE or IgD or a modified variant thereof.
  • the constant domain of the antibody heavy chain may be selected accordingly.
  • the light chain constant domain may be a kappa or lambda constant domain.
  • the antigen binding protein may comprise modifications of all classes e.g. IgG dimers, Fc mutants that no longer bind Fc receptors or mediate C1q binding.
  • the antigen binding protein may also be a chimeric antibody of the type described in WO86/001533 which comprises an antigen binding region and a non-immunoglobulin region.
  • the constant region is selected according to any functionality required e.g. an IgG1 may demonstrate lytic ability through binding to complement and/or will mediate ADCC (antibody dependent cell cytotoxicity) .
  • the antigen binding proteins of the present invention are derived from the murine antibody having the variable regions as described in SEQ ID NO: 10 and SEQ ID NO: 11 or non-murine equivalents thereof, such as rat, human, chimeric or humanised variants thereof, for example they are derived from the antibody having the variable heavy chain sequences as described in SEQ ID NO: 10, and/or the variable light chain sequences as described in SEQ ID NO: 11.
  • an antigen binding protein comprising an isolated heavy chain variable domain selected from any one of the following: SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 13.
  • an antigen binding protein comprising an isolated light chain variable domain selected from any one of the following: SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 15.
  • an antigen binding protein comprising an isolated heavy chain variable domain selected from any one of the following: SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 13 and an isolated light chain variable domain selected from any one of the following: SEQ ID NO: 9, SEQ ID NO: 11 and/or SEQ ID NO: 15.
  • the antigen binding protein of the present invention comprises a heavy chain variable region encoded by SEQ. ID. NO: 20 or SEQ. ID. NO: 22 and a light chain variable region encoded by SEQ. ID. NO: 21 or SEQ. ID. NO: 23
  • polynucleotide encoding an isolated variable heavy chain said polynucleotide comprising SEQ. ID. NO. 28, or SEQ. ID. NO. 29, or SEQ. ID. NO. 30.
  • polynucleotide encoding an isolated variable light chain said polynucleotide comprising SEQ. ID. NO. 31, or SEQ. ID. NO. 32, or SEQ. ID. NO. 33.
  • the antigen binding protein may comprise any one of the variable heavy chains as described herein in combination with any one of the light chains as described herein.
  • the antigen binding protein is an antibody or antigen binding fragment thereof comprising one or more CDR's according to the invention described herein, or one or both of the heavy or light chain variable domains according to the invention described herein.
  • the antigen binding protein binds primate BCMA.
  • the antigen binding protein additionally binds non-human primate BCMA, for example cynomolgus macaque monkey BCMA.
  • the antigen binding protein is selected from the group consisting of a dAb, Fab, Fab', F (ab') . sub. 2, Fv, diabody, triabody, tetrabody, miniantibody, and a minibody.
  • the antigen binding protein is a humanised or chimaeric antibody, in a further aspect the antibody is humanised. In one aspect the antibody is a monoclonal antibody.
  • the antigen binding protein binds to human BCMA with high affinity for example when measured by Biacore the antigen binding protein binds to human BCMA with an affinity of 20 nM or less or an affinity of 15 nM or less or an affinity of 5 nM or less or an affinity of 1000 pM or less or an affinity of 500 pM or less or an affinity of 400 pM or less, or 300 pM or less or for example about 120 pM.
  • the antigen binding protein binds to human BCMA when measured by Biacore of between about 100 pM and about 500 pM or between about 100 pM and about 400 pM, or between about 100 pM and about 300 pM.
  • the antigen binding protein binds BCMA with an affinity of less than 150 pm.
  • this is measured by Biacore, for example as set out in Example 4.
  • the antigen binding protein binds to human BCMA and neutralises the binding of the ligands BAFF and/or APRIL to the BCMA receptor in a cell neutralisation assay wherein the antigen binding protein has an 1050 of between about 1 nM and about 500 nM, or between about 1 nM and about 100 nM, or between about 1 nM and about 50 nM, or between about 1 nM and about 25 nM, or between about 5 nM and about 15 nM.
  • the antigen binding protein binds BCMA and neutralises BCMA in a cell neutralisation assay wherein the antigen binding protein has an 1050 of about 10 nM.
  • the antigen binding proteins for example antibodies of the present invention may be produced by transfection of a host cell with an expression vector comprising the coding sequence for the antigen binding protein of the invention.
  • An expression vector or recombinant plasmid is produced by placing these coding sequences for the antigen binding protein in operative association with conventional regulatory control sequences capable of controlling the replication and expression in, and/or secretion from, a host cell.
  • Regulatory sequences include promoter sequences, e.g., CMV promoter, and signal sequences which can be derived from other known antibodies.
  • a second expression vector can be produced having a DNA sequence which encodes a complementary antigen binding protein light or heavy chain.
  • this second expression vector is identical to the first except insofar as the coding sequences and selectable markers are concerned, so to ensure as far as possible that each polypeptide chain is functionally expressed.
  • the heavy and light chain coding sequences for the antigen binding protein may reside on a single vector.
  • a selected host cell is co-transfected by conventional techniques with both the first and second vectors (or simply transfected by a single vector) to create the transfected host cell of the invention comprising both the recombinant or synthetic light and heavy chains.
  • the transfected cell is then cultured by conventional techniques to produce the engineered antigen binding protein of the invention.
  • the antigen binding protein which includes the association of both the recombinant heavy chain and/or light chain is screened from culture by appropriate assay, such as ELISA or RIA. Similar conventional techniques may be employed to construct other antigen binding proteins.
  • Suitable vectors for the cloning and subcloning steps employed in the methods and construction of the compositions of this invention may be selected by one of skill in the art.
  • the conventional pUC series of cloning vectors may be used.
  • One vector, pUC19 is commercially available from supply houses, such as AmershamBioscience (Buckinghamshire, United Kingdom) or GenScript (Nanjing, China) .
  • any vector which is capable of replicating readily has an abundance of cloning sites and selectable genes (e.g., antibiotic resistance) , and is easily manipulated may be used for cloning.
  • the selection of the cloning vector is not a limiting factor in this invention.
  • the expression vectors may also be characterized by genes suitable for amplifying expression of the heterologous DNA sequences, e.g., the mammalian dihydrofolate reductase gene (DHFR) .
  • Other vector sequences include a poly A signal sequence, such as from bovine growth hormone (BGH) and the betaglobin promoter sequence (betaglopro) .
  • BGH bovine growth hormone
  • betaglopro betaglobin promoter sequence
  • replicons e.g. replicons, selection genes, enhancers, promoters, signal sequences and the like
  • selection genes e.g. replicons, selection genes, enhancers, promoters, signal sequences and the like
  • Other appropriate expression vectors of which numerous types are known in the art for mammalian, bacterial, insect, yeast, and fungal expression may also be selected for this purpose.
  • the present invention also encompasses a cell line transfected with a recombinant plasmid containing the coding sequences of the antigen binding proteins of the present invention.
  • Host cells useful for the cloning and other manipulations of these cloning vectors are also conventional. However, cells from various strains of E. Coli may be used for replication of the cloning vectors and other steps in the construction of antigen binding proteins of this invention.
  • Suitable host cells or cell lines for the expression of the antigen binding proteins of the invention include mammalian cells such as NS0, Sp2/0, CHO (e.g. DG44) , COS, HEK, a fibroblast cell (e.g., 3T3) , and myeloma cells, for example it may be expressed in a CHO or a myeloma cell.
  • mammalian cells such as NS0, Sp2/0, CHO (e.g. DG44) , COS, HEK, a fibroblast cell (e.g., 3T3)
  • myeloma cells for example it may be expressed in a CHO or a myeloma cell.
  • Human cells may be used, thus enabling the molecule to be modified with human glycosylation patterns.
  • eukaryotic cell lines may be employed.
  • suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Sambrook et al., (1989) . Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  • Bacterial cells may prove useful as host cells suitable for the expression of the recombinant Fabs or other embodiments of the present invention (see, e.g., Pluckthun, A., Immunol. Rev., 130: 151-188 (1992) ) .
  • any recombinant Fab produced in a bacterial cell would have to be screened for retention of antigen binding ability.
  • the molecule expressed by the bacterial cell was produced in a properly folded form, that bacterial cell would be a desirable host, or in alternative embodiments the molecule may express in the bacterial host and then be subsequently re-folded.
  • E. Coli used for expression are well-known as host cells in the field of biotechnology.
  • Various strains of B. Subtilis, Streptomyces, other bacilli and the like may also be employed in this method.
  • strains of yeast cells known to those skilled in the art are also available as host cells, as well as insect cells, e.g. Drosophila and Lepidoptera and viral expression systems. See, e.g. Miller et al., Genetic Engineering, 8: 277-298, Plenum Press (1986) and McGuire, S. et al, Trends Genet. (2004) 20, 384-391 and references cited therein.
  • the general methods by which the vectors may be constructed, the transfection methods required to produce the host cells of the invention, and culture methods necessary to produce the antigen binding protein of the invention from such host cell may all be conventional techniques.
  • the culture method of the present invention is a serum-free culture method, usually by culturing cells serum-free in suspension.
  • the antigen binding proteins of the invention may be purified from the cell culture contents according to standard procedures of the art, including ammonium precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Such techniques are within the skill of the art and do not limit this invention. For example, preparations of altered antibodies are described in WO 99/058679 and WO 96/016990.
  • Yet another method of expression of the antigen binding proteins may utilize expression in a transgenic animal, such as described in U.S. Pat. No. 4,873,316. This relates to an expression system using the animals casein promoter which when transgenically incorporated into a mammal permits the female to produce the desired recombinant protein in its milk.
  • a method of producing an antibody of the invention comprises the step of culturing a host cell transformed or transfected with a vector encoding the light and/or heavy chain of the antibody of the invention and recovering the antibody thereby produced.
  • a method of producing an anti-BCMA antibody of the present invention which binds to and neutralises the activity of human BCMA comprises the steps of; providing a first vector encoding a heavy chain of the antibody; providing a second vector encoding a light chain of the antibody; transforming a mammalian host cell (e.g. CHO) with said first and second vectors; culturing the host cell of step (c) under conditions conducive to the secretion of the antibody from said host cell into said culture media; recovering the secreted antibody of step (d) .
  • a mammalian host cell e.g. CHO
  • the antibody is then examined for in vitro activity by use of an appropriate assay.
  • an appropriate assay Presently conventional ELISA assay formats are employed to assess qualitative and quantitative binding of the antibody to BCMA. Additionally, other in vitro assays may also be used to verify neutralizing efficacy prior to subsequent human clinical studies performed to evaluate the persistence of the antibody in the body despite the usual clearance mechanisms.
  • the dose and duration of treatment relates to the relative duration of the molecules (the antibody and the antibody-drug conjugate) of the present invention in the human circulation, and can be adjusted by one of skill in the art depending upon the condition being treated and the general health of the patient. It is envisaged that repeated dosing (e.g. once a week or once every two weeks or once every 3 weeksor once every 4 weeks) over an extended time period (e.g. four to six months) maybe required to achieve maximal therapeutic efficacy.
  • repeated dosing e.g. once a week or once every two weeks or once every 3 weeksor once every 4 weeks
  • an extended time period e.g. four to six months
  • a recombinant transformed, transfected or transduced host cell comprising at least one expression cassette, for example where the expression cassette comprises a polynucleotide encoding a heavy chain of an antigen binding protein according to the invention described herein and further comprises a polynucleotide encoding a light chain of an antigen binding protein according to the invention described herein or where there are two expression cassettes and the 1. sup. st encodes the light chain and the second encodes the heavy chain.
  • the first expression cassette comprises a polynucleotide encoding a heavy chain of an antigen binding protein comprising a constant region or antigen binding fragment thereof which is linked to a constant region according to the invention described herein and further comprises a second cassette comprising a polynucleotide encoding a light chain of an antigen binding protein comprising a constant region or antigen binding fragment thereof which is linked to a constant region according to the invention described herein for example the first expression cassette comprises a polynucleotide encoding a heavy chain selected from SEQ. ID. NO: 18, or SEQ. ID. NO: 25 and a second expression cassette comprising a polynucleotide encoding a light chain selected from SEQ. ID. NO: 19 or SEQ. ID. NO: 27.
  • a stably transformed host cell comprising a vector comprising one or more expression cassettes encoding a heavy chain and/or a light chain of the antibody comprising a constant region or antigen binding fragment thereof which is linked to a constant region as described herein.
  • host cells may comprise a first vector encoding the light chain and a second vector encoding the heavy chain, for example the first vector encodes a heavy chain selected from SEQ. ID. NO: 18, or SEQ. ID. NO: 25 and a second vector encoding a light chain for example the light chain of SEQ ID NO: 19 or SEQ. ID. NO: 27.
  • the first vector encodes a heavy chain selected from SEQ. ID. NO: 18 and a second vector encoding a light chain for example the light chain of SEQ ID NO: 19.
  • Examples of such cell lines include CHO or NS0.
  • a method for the production of an antibody comprising a constant region or antigen binding fragment thereof which is linked to a constant region comprises the step of culturing a host cell in a culture media, for example serum-free culture media.
  • composition comprising an antigen binding protein and a pharmaceutically acceptable carrier.
  • kit-of-parts comprising the composition according to the invention described herein described together with instructions for use.
  • the mode of administration of the therapeutic agent of the invention may be any suitable route which delivers the agent to the host.
  • the antigen binding proteins, and pharmaceutical compositions of the invention are particularly useful for parenteral administration, i.e., subcutaneously (s.c. ) , intrathecally, intraperitoneally, intramuscularly (i.m. ) or intravenously (i.v. ) .
  • the antigen binding proteins of the present invention are administered intravenously or subcutaneously.
  • Therapeutic agents of the invention may be prepared as pharmaceutical compositions containing an effective amount of the antigen binding protein of the invention as an active ingredient in a pharmaceutically acceptable carrier.
  • the prophylactic agent of the invention is an aqueous suspension or solution containing the antigen binding protein in a form ready for injection.
  • the suspension or solution is buffered at physiological pH.
  • the compositions for parenteral administration will comprise a solution of the antigen binding protein of the invention or a cocktail thereof dissolved in a pharmaceutically acceptable carrier.
  • the carrier is an aqueous carrier.
  • a variety of aqueous carriers may be employed, e.g., 0.9%saline, 0.3%glycine, and the like. These solutions may be made sterile and generally free of particulate matter.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc.
  • concentration of the antigen binding protein of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1%to as much as about 15 or 20%by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected.
  • a pharmaceutical composition of the invention for intravenous infusion could be made up to contain about 250 ml of sterile Ringer's solution, and about 1 to about 30 or 5 mg to about 25 mg of an antigen binding protein of the invention per ml of Ringer's solution.
  • Actual methods for preparing parenterally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15. sup. th ed., Mack Publishing Company, Easton, PA, USA.
  • For the preparation of intravenously administrable antigen binding protein formulations of the invention see Parkins D. and Lasmar U. "The formulation of Biopharmaceutical products" , Pharm. Sci. Tech.
  • the antibody of the invention when in a pharmaceutical preparation, is present in unit dose forms.
  • the appropriate therapeutically effective dose will be determined readily by those of skill in the art. Suitable doses may be calculated for patients according to their weight, for example suitable doses may be in the range of about 0.1 to about 200 mg/kg, for example about 1 to about 20 mg/kg, for example about 10 to about 20 mg/kg or for example about 1 to about 15 mg/kg, for example about 5 to about 15 mg/kg.
  • suitable doses may be within the range of about 0.1 to about 2000 mg, for example about 0.1 to about 500 mg, for example about 500 mg, for example about 0.1 to about 150 mg, or about 0.1 to about 80 mg, or about 0.1 to about 60 mg, or about 0.1 to about 40 mg, or for example about 1 to about 100 mg, or about 1 to about 50 mg, of an antigen binding protein of this invention, which may be administered parenterally, for example subcutaneously, intravenously or intramuscularly. Such dose may, if necessary, be repeated at appropriate time intervals selected as appropriate by a physician.
  • antigen binding proteins described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-known peroxidise and reconstitution techniques can be employed.
  • an antigen binding protein as herein described for use in a medicament.
  • an antigen binding protein according to the invention as herein described for use in the treatment of rheumatoid arthitis, Type 1 Diabetes Mellitus, multiple sclerosis or psoriasis wherein said method comprises the step of administering to said patient a therapeutically effective amount of the antigen binding protein as described herein.
  • methods for treating cancer in a human comprising administering to said human an antigen binding protein that specifically binds to BCMA.
  • the antigen binding protein is part of an immunoconjugate.
  • B-cell disorders can be divided into defects of B-cell development/immunoglobulin production (immunodeficiencies) and excessive/uncontrolled proliferation (lymphomas, leukemias) .
  • B-cell disorder refers to both types of diseases, and methods are provided for treating B-cell disorders with an antigen binding protein.
  • the disease or disorder is selected from the group consisting of Multiple Myeloma (MM) , Chronic Lymphocytic Leukaemia (CLL) , Solitary Plasmacytoma (Bone, Extramedullary) , Waldenstrom's Macroglobulinemia.
  • MM Multiple Myeloma
  • CLL Chronic Lymphocytic Leukaemia
  • Solitary Plasmacytoma Bone, Extramedullary
  • Waldenstrom's Macroglobulinemia is selected from the group consisting of Multiple Myeloma (MM) , Chronic Lymphocytic Leukaemia (CLL) , Solitary Plasmacytoma (Bone, Extramedullary) , Waldenstrom's Macroglobulinemia.
  • the disease is Multiple Myeloma, Smoldering Multiple Myeloma (SMM) or Solitary Plasmacytoma (Bone, Extramedullary) .
  • the disease is Multiple Myeloma.
  • the disease is Systemic lupus erythematosus (SLE)
  • the disease is Idiopathic thrombocytopenic purpura (ITP)
  • the antigen binding protein as described herein for use in the treatment or prophylaxis of diseases and disorders responsive to modulation (such as inhibiting or blocking) of the interaction between BCMA and the ligands BAFF and APRIL.
  • the antigen binding protein as described herein for use in the treatment or prophylaxis of an antibody mediated or plasma cell mediated disease or disorder selected from rheumatoid arthitis, Type 1 Diabeted Mellitus, multiple sclerosis or psoriasis.
  • an antibody mediated or plasma cell mediated disease or disorder selected from Multiple Myeloma (MM) , chronic lymphocytic leukemia (CLL) , Monoclonal gammopathy of undetermined significance (MGUS) , Smoldering multiple myeloma (SMM) , Solitary Plasmacytoma (Bone, Extramedullary) , Waldenstrom's Macroglobulinemia, Primary Amyloidosis (AL) , Heavy chain disease, Systemic lupus erythematosus (SLE) , POEMS syndrome/osteosclerotic myeloma, Type I and II cryoglobulinemia, Light chain deposition disease, Goodpastures syndrome, Idiopathic thrombocytopenic purpura (ITP) , Acute glomerulonephritis, Pemphigus and Pemphigoid disorders and Epi
  • MM Multiple Myeloma
  • CLL chronic lymphocytic leukemia
  • MGUS
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody of the present invention or a functional fragment thereof and a pharmaceutically acceptable carrier for treatment or prophylaxis of rheumatoid arthitis, Type 1 Diabetes Mellitus, multiple sclerosis or psoriasis or an antibody mediated or plasma cell mediated disease or disorder selected from selected from Multiple Myeloma (MM) , chronic lymphocytic leukemia (CLL) , Monoclonal gammopathy of undetermined significance (MGUS) , Smoldering multiple myeloma (SMM) , Solitary Plasmacytoma (Bone, Extramedullary) , Waldenstrom's Macroglobulinemia, Primary Amyloidosis (AL) , Heavy chain disease, Systemic lupus erythematosus (SLE) , POEMS syndrome/osteosclerotic myeloma, Type I and II cryoglobulinemia, Light chain deposition disease, Goodpas
  • a method of treating a human patient afflicted with rheumatoid arthitis, Type 1 Diabetes Mellitus, multiple sclerosis or psoriasis or an antibody mediated or plasma cell mediated disorder or disease which method comprises the step of administering a therapeutically effective amount of the antigen binding protein according to the invention as described herein, for example there is provided a method of treating a human patient afflicted with an antibody mediated or plasma cell mediated disease or disorder selected from
  • MM Multiple Myeloma
  • the BCMA antibody described herein is useful for any therapeutic in which it is desirable to target BCMA, such as adoptive cell transfer (ACT) , bispecific T-cell engagers (BiTEs) , and nanoparticles.
  • the disclosure provides a chimeric antigen receptor (CAR) comprising an antigen binding domain of the BCMA monoclonal antibody described herein linked to a T-cell activation moiety.
  • a “chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide containing an antigen binding domain of an antibody (e.g., a single chain variable fragment (scFv) ) linked to T-cell signaling or T-cell activation moeities.
  • CAR structures have evolved over the last twenty years to most commonly incorporate a single chain variable fragment (scFv) derived from a monoclonal antibody (mAb) and the signaling motif from the TCR chain (referred to as a "first-generation" CAR (see, e.g., Okur, F.V., Brenner, M.K., Methods Mol. Biol., 651: 319-45 (2010) ; and Lee et al., Clin. Cancer. Res., 18 (10) : 2780-2790 (2012) ) .
  • scFv single chain variable fragment
  • mAb monoclonal antibody
  • first-generation CAR
  • second and third generation CARs have been developed, which incorporate one ( “second generation” ) or two ( “third generation” ) costimulatory activating motifs from, for example, CD28, 4-1BB (CD137) , and/or CD134 (OX-40) , which enhance proliferation, cytotoxicity, and persistence in vivo (see, e.g., Finney et al., J. Immunol., 172: 104-13 (2004) ; Altvater et al, Clin Exp Immunol. 144 (3) : 447-57 (2006) ; Chu et al, J Transl Med.
  • the antigen binding domain of the CAR may comprise a whole monoclonal antibody or a monoclonal antibody fragment, as described herein.
  • the antigen binding domain of the CAR may comprise a single chain Fv (scFv) fragment of the anti-BCMA monoclonal antibody.
  • scFv single chain Fv
  • ADC antibody-drug conjugate
  • mAb monoclonal antibody
  • cytotoxic agent generally a small molecule drug with a high systemic toxicity
  • D-L-mAb (I) wherein D, D 1 and D 2 area small molecule cytotoxin or a functional small molecule, in general called payload; L, L 1 and L 2 are a linker; and mAb is an monoclonal antibody.
  • an ADC may comprise a small molecule cytotoxin that has been chemically modified to contain a linker, or a linker is part of payload which is called a traceless linker.
  • the linker is generally used to conjugate the cytotoxin to the antibody, or antigen-binding fragment thereof.
  • the ADC Upon binding to the target antigen on the surface of a cell, the ADC is internalized and trafficked to the lysosome where the cytotoxin is released by either proteolysis of a cleavable linker (e.g., by cathepsin B found in the lysosome) or by proteolytic degradation of the antibody, if attached to the cytotoxin via a non-cleavable linker.
  • the cytotoxin then translocates out of the lysosome and into the cytosol or nucleus, where it can then bind to its target, depending on its mechanism of action.
  • the antibody-drug conjugate described herein may comprise a whole antibody or an antibody fragment.
  • a whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide.
  • Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region.
  • the variable regions of each pair of light and heavy chains form the antigen binding site of an antibody.
  • the VH and VL regions have the same general structure, with each region comprising four framework regions, whose sequences are relatively conserved.
  • the framework regions are connected by three complementarity determining regions (CDRs) .
  • the three CDRs known as CDR1, CDR2, and CDR3, form the "hypervariable region" of an antibody, which is responsible for antigen binding.
  • the ADC may comprise an antigen-binding fragment of an antibody.
  • antibody fragment used interchangeably herein and refer to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen.
  • the antibody fragment may comprise, for example, one or more CDRs, the variable region (or portions thereof) , the constant region (or portions thereof) , or combinations thereof.
  • antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F (ab') 2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (iv) a single chain Fv (scFv) , which is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., VL and VH) joined by a synthetic linker which enables the two domains to be synthesized as a single polypeptide chain (see, e.g., Kabat EA, Wu TT., J Immunol.
  • a diabody which is a dimer of polypeptide chains, wherein each polypeptide chain comprises a VH connected to a VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby driving the pairing between the complementary domains on different VH-VL polypeptide chains to generate a dimeric molecule having two functional antigen binding sites (see, e.g. Hudson PJ, Kortt AA, J Immunol Methods. 1999, 231 (1-2) : 177-89; Holliger P, Winter G. Cancer Immunol Immunother. 1997, 45 (3-4) : 128-30) .
  • the antibody-drug conjugate described herein comprises a monoclonal antibody, or an antigen-binding fragment thereof, directed against B-cell Maturation Antigen (BCMA, also known as CD269) .
  • the monoclonal antibody, or antigen-binding fragment thereof may comprise (a) a heavy chain variable region comprising a complementarity determining region 1 (HCDR1) amino acid sequence of SEQ ID NO: 1, an HCDR2 amino acid sequence of SEQ ID NO: 2, and an HCDR3 amino acid sequence of SEQ ID NO: 3 and (b) a light chain variable region comprising a complementarity determining region 1 (LCDR1) amino acid sequence of SEQ ID NO: 4, an LCDR2 amino acid sequence of SEQ ID NO: 5, and an LCDR3 amino acid sequence of SEQ ID NO: 6.
  • the monoclonal antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9.
  • the monoclonal antibody, or an antigen-binding fragment thereof, directed against BCMA may comprise any suitable binding affinity to BCMA or an epitope thereof.
  • affinity refers to the equilibrium constant for the reversible binding of two agents and is expressed as the dissociation constant (K D ) .
  • K D dissociation constant
  • the affinity of an antibody or antigen-binding fragment thereof for an antigen or epitope of interest can be measured using any method known in the art. Such methods include, for example, fluorescence activated cell sorting (FACS) , surface plasmon resonance (e.g., Biacore TM , ProteOn TM ) , biolayer interferometry (BLI, e.g. Octet) , kinetics exclusion assay (e.g.
  • KinExA TM separable beads (e.g., magnetic beads) , antigen panning, and/or ELISA (see, e.g., J R Crowther, Methods Mol Biol. 2000, 149: III-IV, 1-413) . It is known in the art that the binding affinity of a particular antibody will vary depending on the method that is used to analyze the binding affinity.
  • Affinity of a binding agent to a ligand can be, for example, from about 1 picomolar (pM) to about 1 micromolar (1 ⁇ M) (e.g., from about 1 picomolar (pM) to about 1 nanomolar (nM) , or from about 1 nM to about 1 micromolar ( ⁇ M) ) .
  • the monoclonal antibody or an antigen-binding fragment thereof may bind to BCMA with a Kd less than or equal to 100 nanomolar (e.g., 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, or about 10 nM, or a range defined by any two of the foregoing values) .
  • 100 nanomolar e.g., 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, or about 10 nM, or a range defined by any two of the foregoing values
  • the monoclonal antibody may bind to BCMA with a Kd less than or equal to 10 nanomolar (e.g., about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM, about 0.05 nM, about 0.02 nM, about 0.01 nM, about 0.001 nM, or a range defined by any two of the foregoing values) .
  • a Kd less than or equal to 10 nanomolar (e.g., about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3
  • the monoclonal antibody may bind to BCMA with a Kd less than or equal to 200 pM (e.g., about 190 pM, about 175 pM, about 150 pM, about 125 pM, about 110 pM, about 100 pM, about 90 pM, about 80 pM, about 70pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, about 1 pM, or a range defined by any two of the foregoing values) .
  • 200 pM e.g., about 190 pM, about 175 pM, about 150 pM, about 125 pM, about 110 pM, about 100 pM, about 90 pM, about 80 pM, about 70pM, about 60 pM, about 50 pM, about 40 pM, about 30
  • the affinity of the BCMA antibody or antigen-binding fragment thereof to monomeric BCMA is about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, or a range defined by any two of the foregoing values, for example, about 50 nM to about 70 nM, about 55 nM to about 65 nM, or about 58 nM to about 62 nM.
  • the affinity of the BCMA antibody or antigen-binding fragment thereof to membrane-bound BCMA, as measured by FACS, is less than or equal to 10 nanomolar (e.g., about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM, about 0.05 nM, about 0.02 nM, about 0.01 nM, about 0.001 nM, or a range defined by any two of the foregoing values) .
  • 10 nanomolar e.g., about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM
  • an antigen-binding portion or fragment of a monoclonal antibody can be of any size so long as the portion binds to BCMA.
  • an antigen binding portion or fragment of the monoclonal antibody directed against BCMA desirably comprises between about 5 and 25 amino acids (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 35 ora range defined by any two of the foregoing values) .
  • the antibody-drug conjugate comprises a variable region of an anti-BCMA monoclonal antibody.
  • the ADC may comprise a light chain variable region, a heavy chain variable region, or both a light chain variable region and a heavy chain variable region of an anti-BCMA monoclonal antibody.
  • the ADC comprises a light chain variable region and a heavy chain variable region of an anti-BCMA monoclonal antibody.
  • the monoclonal antibody of the ADC described herein comprises (a) a heavy chain variable region comprising a complementarity determining region 1 (HCDR1) amino acid sequence of TSFLIHW (SEQ ID NO: 1) , an HCDR2 amino acid sequence of FIIPGNGGTKYNQKFQ (SEQ ID NO: 2) , and an HCDR3 amino acid sequence of YDGSFEGYFDV (SEQ ID NO: 3) and (b) a light chain variable region comprising a complementarity determining region 1 (LCDR1) amino acid sequence of SSQSLVHSDGNTYLH (SEQ ID NO: 4) , an LCDR2 amino acid sequence of KVSNRDS (SEQ ID NO: 5) , and an LCDR3 amino acid sequence of SQSTHWPWT (SEQ ID NO: 6) .
  • the monoclonal antibody of the ADC described herein may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and/
  • the BCMA monoclonal antibody, or antigen-binding fragment thereof may be conjugated to a cytotoxin using any suitable method known in the art, including site-specific or non-site specific conjugation methods.
  • Conventional conjugation strategies for antibodies typically rely on randomly (i.e., non-specifically) conjugating the payload to the antibody, antigen-binding fragment thereof, through lysines or cysteines.
  • the antibody or antigen-binding fragment thereof is randomly conjugated to a cytotoxic agent, for example, by partial reduction of the antibody or antibody fragment, followed by reaction with a desired agent with or without a linker moiety attached.
  • the antibody or antigen-binding fragment thereof may be reduced using dithiothreitol (DTT) , TCEP, thiolethenol or a similar reducing agent.
  • DTT dithiothreitol
  • TCEP TCEP
  • thiolethenol or a similar reducing agent.
  • the cytotoxic agent, with or without a linker moiety attached thereto, can then be added at a molar excess to the reduced antibody or antibody fragment in the presence of dimethyl sulfoxide (DMSO) , or DMA. After conjugation, excess free cysteine may be added to quench unreacted agent.
  • DMSO dimethyl sulfoxide
  • DMA dimethyl sulfoxide
  • excess free cysteine may be added to quench unreacted agent.
  • the cytotoxic agent, with or without a linker moiety having an amino-reactivable, or phenol-reactivable, or the others reactivable group e.g.
  • NHS, PFP thereto, can be added directly at a molar excess to the antibody or antibody fragment in the presence of DMSO, or DMA to form a conjugate.
  • the reaction mixture may then be purified through chromatography or buffer-exchanged into phosphate buffered saline (PBS) .
  • PBS phosphate buffered saline
  • cytotoxin and cytotoxic agent refer to any molecule that inhibits or prevents the function of cells and/or causes destruction of cells (cell death) , and/or exerts anti-proliferative effects.
  • a cytotoxin or cytotoxic agent of an ADC also is referred to in the art as the "payload" of the ADC.
  • a number of classes of cytotoxic agents are known in the art to have potential utility in ADC molecules and can be used in the ADC described herein.
  • Such classes of cytotoxic agents include, for example, anti-microtubule agents (e.g., tubulysins, auristatins and maytansinoids) , DNA minor groove binders (e.g.
  • pyrrolobenzodiazepines PBDs or indolinobenzodiazepines (IGN)
  • RNA polymerase II inhibitors e.g., amatoxins
  • inhibitor of DNA topoisomerase I e.g., camptothecins
  • DNA alkylating agents e.g., duocarmycin, CC-1065, pyrrolobenzodiazepineor indolinobenzodiazepinepseudodimers
  • cytotoxic agents examples include, but are not limited to, tubulysins, amanitins, auristatins, calicheamicin, camptothecins, daunomycins, doxorubicins, duocarmycins, dolastatins, enediynes, lexitropsins, taxanes, puromycins, maytansinoids, vinca alkaloids, and pyrrolobenzodiazepines (PBDs) .
  • tubulysins examples include, but are not limited to, tubulysins, amanitins, auristatins, calicheamicin, camptothecins, daunomycins, doxorubicins, duocarmycins, dolastatins, enediynes, lexitropsins, taxanes, puromycins, maytansinoids, vinca alkaloids, and pyrrolobenzodiazepines (PBDs) .
  • the cytotoxic agent may be, for example tubulysins, auristatins (AFP, MMAF, MMAE, AEB, AEVB, E) , paclitaxels, docetaxels, CC-1065 (ducarmysin, DC1, DC4, CBI-dimers) , camptothecins (SN-38, topotecans) , morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, chalicheamicin, maytansine (DM1, DM4, DM21) , vinblastine, methotrexate, netropsin, or derivatives or analogs thereof. Cytotoxins suitable for use in ADCs are also described in, for example, International Patent Application Publication No. PCT/CN2021/128453.
  • chemotherapeutic agent or a functional compound can also be conjugated to the BCMA antibody of this invention.
  • a chemotherapeutic agent or a functional compound is selected from the group consisting of:
  • an alkylating agent selected from the group consisting ofnitrogen mustards: chlorambucil, chlornaphazine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, mitobronitol, melphalan, mitolactol, pipobroman, novembichin, phenesterine, prednimustine, thiotepa, trofosfamide, uracil mustard; CC-1065 andadozelesin, carzelesin, bizelesinor their synthetic analogues; duocarmycinandits synthetic analogues, KW-2189, CBI-TMI, or CBI dimers; benzodiazepine dimers orpyrrolobenzodiazepine (PBD) dimers, tomaymycindimers, indolinobenzodiazepinedimers, imi
  • a plant alkaloid selected from the group consisting ofVinca alkaloids: comprising vincristine, vinblastine, vindesine, vinorelbine, and navelbin; Taxoids: comprisingpaclitaxel, docetaxol and their analogs, Maytansinoids comprising DM1, DM2, DM3, DM4, DM5, DM6, DM7, maytansine, ansamitocinsand their analogs, cryptophycins (including the group consisting of cryptophycin 1 and cryptophycin 8) ; epothilones, eleutherobin, discodermolide, bryostatins, dolostatins, auristatins, tubulysins, cephalostatins; pancratistatin; erbulins, a sarcodictyin; spongistatin;
  • a DNA Topoisomerase Inhibitor selected from the groups ofEpipodophyllins: comprising 9-aminocamptothecin, camptothecin, crisnatol, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acids (or retinols) , teniposide, topotecan, 9-nitrocamptothecin or RFS 2000; and mitomycins and their analogs;
  • An antimetabolite selected from the group consisting of ⁇ [Anti-folate: (DHFR inhibitors: comprising methotrexate, trimetrexate, denopterin, pteropterin, aminopterin (4-aminopteroic acid) or folic acid analogues) ; IMP dehydrogenase Inhibitors: (comprising mycophenolic acid, tiazofurin, ribavirin, EICAR) ; Ribonucleotide reductase Inhibitors: (comprisinghydroxyurea, deferoxamine) ] ; [pyrimidine analogs: Uracil analogs: (comprising ancitabine, azacitidine, 6-azauridine, capecitabine (Xeloda) , carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-fluorouracil, floxuridine, ratitrexed (DHFR inhibitors:
  • a hormonal therapy selected from the group consisting of ⁇ Receptor antagonists: [Anti-estrogen: (comprising megestrol, raloxifene, tamoxifen) ; LHRH agonists: (comprisinggoscrclin, leuprolide acetate) ; Anti-androgens: (comprising bicalutamide, flutamide, calusterone, dromostanolone propionate, epitiostanol, goserelin, leuprolide, mepitiostane, nilutamide, testolactone, trilostane and other androgens inhibitors) ] ; Retinoids/Deltoids: [Vitamin D3 analogs: (comprising CB 1093, EB 1089 KH 1060, cholecalciferol, ergocalciferol) ; Photodynamic therapies: (comprising verteporfin, phthalo
  • a kinase inhibitor selected from the group consisting ofBIBW 2992 (anti-EGFR/Erb2) , imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib.
  • vandetanib E7080 (anti-VEGFR2) , mubritinib, ponatinib (AP24534) , bafetinib (INNO-406) , bosutinib (SKI-606) , cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, Trastuzumab, Ranibizumab, Panitumumab, ispinesib;
  • a poly (ADP-ribose) polymerase (PARP) inhibitors selected from the group consisting ofolaparib, niraparib, iniparib, talazoparib, veliparib, CEP 9722 (Cephalon’s) , E7016 (Eisai's) , BGB-290 (BeiGene’s) , or3-aminobenzamide.
  • PARP poly (ADP-ribose) polymerase
  • An antibiotic selected from the group consisting ofan enediyne antibiotic (selected from the group consisting of calicheamicin, calicheamicin ⁇ 1, ⁇ 1, ⁇ 1 or ⁇ 1; dynemicin, including dynemicin A and deoxydynemicin; esperamicin, kedarcidin, C-1027, maduropeptin, orneocarzinostatin chromophore and related chromoprotein enediyne antibioticchromomophores) , aclacinomycins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin; chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubic
  • a polyketide acetogenin
  • bullatacin and bullatacinone gemcitabine, epoxomicinsandcarfilzomib, bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allovectin-7, Xegeva, Provenge, Yervoy, Isoprenylation inhibitors and Lovastatin, Dopaminergic neurotoxins and1-methyl-4-phenylpyridinium ion, Cell cycle inhibitors (selected fromstaurosporine) , Actinomycins (comprising Actinomycin D, dactinomycin) , amanitins, Bleomycins (comprising bleomycin A2, bleomycin B2, peplomycin) , Anthracyclines (comprising daunor
  • An anti-autoimmune disease agent cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (including the group consisting of amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, fluocortolone danazol, dexamethasone, Triamcinolone acetonide, beclometasone dipropionate) , DHEA, enanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mofetil, mycophenylate, prednisone, sirolimus, tacrolimus.
  • corticosteroids including the group consisting of amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticas
  • An anti-infectious disease agents comprising:
  • Aminoglycosides amikacin, astromicin, gentamicin (netilmicin, sisomicin, isepamicin) , hygromycin B, kanamycin (amikacin, arbekacin, bekanamycin, dibekacin, tobramycin) , neomycin (framycetin, paromomycin, ribostamycin) , netilmicin, spectinomycin, streptomycin, tobramycin, verdamicin;
  • Amphenicols azidamfenicol, chloramphenicol, florfenicol, thiamphenicol;
  • Ansamycins geldanamycin, herbimycin;
  • Carbapenems biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem;
  • Cephems carbacephem (loracarbef) , cefacetrile, cefaclor, cefradine, cefadroxil, cefalonium, cefaloridine, cefalotin or cefalothin, cefalexin, cefaloglycin, cefamandole, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin, cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefozo
  • Glycopeptides bleomycin, vancomycin (oritavancin, telavancin) , teicoplanin (dalbavancin) , ramoplanin;
  • Glycylcyclines tigecycline
  • ⁇ -Lactamase inhibitors penam (sulbactam, tazobactam) , clavam (clavulanic acid) ;
  • Lincosamides clindamycin, lincomycin
  • Lipopeptides daptomycin, A54145, calcium-dependent antibiotics (CDA) ;
  • Macrolides azithromycin, cethromycin, clarithromycin, dirithromycin, erythromycin, flurithromycin, josamycin, ketolide (telithromycin, cethromycin) , midecamycin, miocamycin, oleandomycin, rifamycins (rifampicin, rifampin, rifabutin, rifapentine) , rokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506) , troleandomycin, telithromycin;
  • Penicillins amoxicillin, ampicillin, pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin, azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethylpenicillin, clometocillin, procaine benzylpenicillin, carbenicillin (carindacillin) , cloxacillin, dicloxacillin, epicillin, flucloxacillin, mecillinam (pivmecillinam) , mezlocillin, meticillin, nafcillin, oxacillin, penamecillin, penicillin, pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin, sulbenicillin, temocillin, ticarcillin;
  • Polypeptides bacitracin, colistin, polymyxin B;
  • Streptogramins pristinamycin, quinupristin/dalfopristin;
  • Sulfonamides mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole (co-trimoxazole) ;
  • Steroid antibacterials selected fromfusidic acid;
  • Tetracyclines doxycycline, chlortetracycline, clomocycline, demeclocycline, lymecycline, meclocycline, metacycline, minocycline, oxytetracycline, penimepicycline, rolitetracycline, tetracycline, glycylcyclines (including tigecycline) ;
  • antibiotics selected from the group consisting of annonacin, arsphenamine, bactoprenol inhibitors (Bacitracin) , DADAL/AR inhibitors (cycloserine) , dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitors (fosfomycin) , nitrofurantoin, paclitaxel, platensimycin, pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampin) , tazobactam tinidazole, uvaricin;
  • Entry/fusion inhibitors aplaviroc, maraviroc, vicriviroc, gp41 (enfuvirtide) , PRO 140, CD4 (ibalizumab) ;
  • Integrase inhibitors raltegravir, elvitegravir, globoidnan A;
  • Maturation inhibitors bevirimat, becon
  • Neuraminidase inhibitors oseltamivir, zanamivir, peramivir;
  • Nucleosides &nucleotides abacavir, aciclovir, adefovir, amdoxovir, apricitabine, brivudine, cidofovir, clevudine, dexelvucitabine, didanosine (ddI) , elvucitabine, emtricitabine (FTC) , entecavir, famciclovir, fluorouracil (5-FU) , 3’-fluoro-substituted 2’, 3’-dideoxynucleoside analogues (including the group consisting of3’-fluoro-2’, 3’-dideoxythymidine (FLT) and 3’-fluoro-2’, 3’-dideoxyguanosine (FLG) , fomivirsen, ganciclovir, idoxuridine, lamivudine (3TC) , l-nu
  • Non-nucleosides amantadine, ateviridine, capravirine, diarylpyrimidines (etravirine, rilpivirine) , delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphonoformic acid) , imiquimod, interferon alfa, loviride, lodenosine, methisazone, nevirapine, NOV-205, peginterferon alfa, podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848) , tromantadine;
  • Protease inhibitors amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950) , tipranavir;
  • anti-virus drugs abzyme, arbidol, calanolide a, ceragenin, cyanovirin-n, diarylpyrimidines, epigallocatechin gallate (EGCG) , foscarnet, griffithsin, taribavirin (viramidine) , hydroxyurea, KP-1461, miltefosine, pleconaril, portmanteau inhibitors, ribavirin, seliciclib.
  • EGCG epigallocatechin gallate
  • griffithsin taribavirin (viramidine)
  • KP-1461 miltefosine
  • pleconaril portmanteau inhibitors
  • ribavirin seliciclib.
  • a radioisotope that can be selected from the group consisting of (radionuclides) 3 H, 11 C, 14 C, 18 F, 32 P, 35 S, 64 Cu, 68 Ga, 86 Y, 99 Tc, 111 In, 123 I, 124 I, 125 I, 131 I, 133 Xe, 177 Lu, 211 At, or 213 Bi.
  • a chromophore molecule whichis capable of absorbing UV light, florescent light, IR light, near IR light, visual light;
  • Non-protein organic fluorophores selected from: Xanthene derivatives (comprising fluorescein, rhodamine, Oregon green, eosin, and Texas red) ; Cyanine derivatives: (comprising cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine) ; Squaraine derivatives and ring-substituted squaraines, including Seta, Se
  • the cell-binding ligands or receptor agonists which can be selected from: Folate derivatives; Glutamic acid urea derivatives; Somatostatin and its analogs (selected from the group consisting of octreotide (Sandostatin) and lanreotide (Somatuline) ) ; Aromatic sulfonamides; Pituitary adenylate cyclase activating peptides (PACAP) (PAC1) ; Vasoactive intestinal peptides (VIP/PACAP) (VPAC1, VPAC2) ; Melanocyte-stimulating hormones ( ⁇ -MSH) ; Cholecystokinins (CCK) /gastrin receptor agonists; Bombesins (selected from the group consisting ofPyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH 2 ) /
  • the drug D can be polyalkylene glycols that are used for extending the half-life of the cell-binding antibody, or antibodymolecule when administered to a mammal.
  • Polyalkylene glycols include, but are not limited to, poly (ethylene glycols) (PEGs) , poly (propylene glycol) and copolymers of ethylene oxide and propylene oxide; particularly preferred are PEGs, and more particularly preferred are monofunctionally activated hydroxyPEGs (e.g., hydroxyl PEGs activated at a single terminus, including reactive esters of hydroxyPEG-monocarboxylic acids, hydroxyPEG-monoaldehydes, hydroxyPEG-monoamines, hydroxyPEG-monohydrazides, hydroxyPEG-monocarbazates, hydroxyl PEG-monoiodoacetamides, hydroxyl PEG-monomaleimides, hydroxyl PEG-monoorthopyridyl disulfides,
  • the polyalkylene glycol has a molecular weight of from about 10 Daltons to about 200 kDa, preferably about 88 Da to about 40 kDa; two branches each with a molecular weight of about 88 Da to about 40 kDa; and more preferably two branches, each of about 88 Da to about 20 kDa.
  • the polyalkylene glycol is poly (ethylene) glycol and has a molecular weight of about 10 kDa; about 20 kDa, or about 40 kDa.
  • the PEG is a PEG 10 kDa (linear or branched) , a PEG 20 kDa (linear or branched) , or a PEG 40 kDa (linear or branched) .
  • a number of USpatents have disclosed the preparation of linear or branched “non-antigenic” PEG polymers and derivatives or conjugates thereof, e.g., U.S. Pat. Nos.
  • D is more preferably a potent cytotoxic agent, selected from a tubulysin and its analogs, a maytansinoid and its analogs, a taxanoid (taxane) and its analogs, a CC-1065 and its analogs, a daunorubicin or doxorubicin and its analogs, an amatoxin and its analogs, a benzodiazepine dimer (e.g., dimers of pyrrolobenzodiazepine (PBD) , tomaymycin, anthramycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinobenzo-diazepines) and their analogs, a calicheamicin and the enediyne antibiotic and their analogs, an actinomycin and its analogs, an azaserine and its analogs, a bleomycin and its analogs, an epirubicin and its analogs,
  • Tubulysin and its analogs are well known in the art and can be isolated from natural sources according to known methods or prepared synthetically according to known methods (e.g. Balasubramanian, R., et al. J. Med. Chem., 2009, 52, 238–40; Wipf, P., et al. Org. Lett., 2004, 6, 4057–60; Pando, O., et al. J. Am. Chem. Soc., 2011, 133, 7692–5; Reddy, J.A., et al. Mol. Pharmaceutics, 2009, 6, 1518–25; Raghavan, B., et al. J. Med.
  • Tubulysin analog having the following formula (IV) :
  • isalinkagesite that either one or two of them can link to L 1 and/or L 2 independently; when two of link to both L 1 and L 2 , R 1 and R 2 , or Z 2 and Z 3 are preferably the dual linkage sites;
  • R 1 , R 1’ , R 2 , R 3 , andR 4 are independently H, C 1 ⁇ C 8 alkyl; C 2 ⁇ C 8 heteroalkyl, or heterocyclic; C 3 ⁇ C 8 aryl, Ar-alkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbocyclic, or alkylcarbonyl; or R 1 R 2 , R 1 R 3 , R 2 R 3 , R 3 R 4 , or R 5 R 6 form a 3 ⁇ 7 membered carbocyclic, cycloalkyl, heterocyclic, heterocycloalkyl, aromatic or heteroaromatic ring system; R 1 and R 2 can be independently absent when they link to L 1 or L 2 independently or simultaneously, Y 1 is N or CH;
  • R 5 , R 6 , R 8 , R 10 andR 11 are independently H, or C 1 ⁇ C 4 alkyl orheteroalkyl;
  • X 1 is O, S, S-S, NH, CH 2 or NR 14 ;
  • R 13 and R 14 are independentlyC 1 ⁇ C 8 alkyl, heteroalkyl; C 2 -C 8 of alkenyl, alkynyl, heteroalkyl, heterocycloalkyl; C 3 -C 8 of aryl, Ar-alkyl;
  • R 15 , R 16 and R 17 are independently H, C 1 ⁇ C 8 alkyl, heteroalkyl; C 2 -C 8 of alkenyl, alkynyl, heteroalkyl, heterocycloalkyl; C 3 -C 8 of aryl, Ar-alkyl, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, alkylcarbonyl, or Na + , K + , Cs + , Li + , Ca 2+ , Mg + , Zn 2+ , N + (R 1 ) (R 2 ) (R 3 ) (R 4 ) , HN + (C 2 H 5 OH) 3 salt;
  • R 20 is H; C 1 -C 8 of linear or branched alkyl or heteroalkyl; C 2 -C 8 of linear or branched alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C 3 -C 8 linear or branched of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate (-C (O) OR 17 ) , carbamate (-C (O) NR 17 R 18 ) ; or 1-8 carbon atoms of carboxylate, esters, ether, or amide; or 1 ⁇ 8 amino acids; or polyethyleneoxy unit of formula (OCH 2 CH 2 ) p or (OCH 2 CH (CH 3 ) ) p , wherein p is an integer from 0 to about 1000; or R 20 is absent and the oxygene forms a ketone, or combination above thereof
  • Calicheamicins and their related enediyne antibiotics that are described in: Nicolaou, K. C. et al, Science 1992, 256, 1172-1178; Proc. Natl. Acad. Sci USA. 1993, 90, 5881-8) , U.S. Patent Nos. 4,970,198; 5,053,394; 5,108,912; 5,264,586; 5,384,412; 5,606,040; 5,712,374; 5,714,586; 5,739,116; 5,770,701; 5,770,710; 5,773,001; 5,877,296; 6,015,562; 6,124,310; 8,153,768.
  • Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, uncialamicin, dynemicin, and their derivatives.
  • the structure of calicheamicins is preferred the following formula:
  • Geldanamycins are benzoquinone ansamycin antibiotic that bind to Hsp90 (Heat Shock Protein 90) and have been used antitumor drugs.
  • exemplary geldanamycins include, but are not limited to, 17-AAG (17-N-Allylamino-17-Demethoxygeldanamycin) and 17-DMAG (17-Dimethylaminoethylamino-17-demethoxygeldanamycin) .
  • Maytansines or their derivatives maytansinoids inhibit cell proliferation by inhibiting the mcirotubules formation during mitosis through inhibition of polymerization of tubulin. See Remillard et al., Science 189: 1002-1005 (1975) .
  • Exemplary maytansines and maytansinoids include, but are not limited to, mertansines (DM1, DM4) , maytansinol and its derivatives as well as ansamitocin. Maytansinoidsare described in U.S. Patent Nos.
  • camptothecin and its derivatives, which aretopoisomerase inhibitors to prevent DNA re-ligation and therefore to causes DNA damage resulting in apoptosis, are described in: Shang, X.F. et al, Med Res Rev. 2018, 38 (3) : 775-828; Botella, P. and Rivero-Buceta, E. J Control Release. 2017, 247: 28-54; Martino, E. et al, Bioorg Med Chem Lett. 2017, 27 (4) : 701-707; Lu, A., et al, Acta Pharmacol Sin 2007, 28 (2) : 307–314.
  • Camptothecin CPT
  • R 1 , R 2 and R 4 are independently selected from H, F, Cl, Br, CN, NO 2 , C 1 ⁇ C 8 alkyl; O-C 1 ⁇ C 8 alkyl; NH-C 1 ⁇ C 8 alkyl; C 2 -C 8 of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C 3 -C 8 of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or 2-8 carbon atoms of esters, ether, amide, carbonate, urea, or carbamate; R 3 is H, OH, NH 2 , C 1
  • camptothecins are preferred the following formula:
  • P 1 is H, OH, NH 2 , COOH, C (O) NH 2 , OCH 2 OP (O) (OR 18 ) 2 , OC (O) OP (O) (OR 18 ) 2 , OPO (OR 18 ) 2 , NHPO (OR 18 ) 2 , OC (O) R 18 , OP (O) (OR 18 ) OP (O) (OR 18 ) 2 , OC (O) NHR 18 , OC (O) N (C 2 H 4 ) 2 NCH 3 , OSO 2 (OR 18 ) , O- (C 4 -C 12- glycoside) , OC (O) N (C 2 H 4 )
  • Combretastatins are natural phenols with vascular disruption properties in tumors.
  • Exemplary combretastatins and their derivatives include, but are not limited to, combretastatin A-4 (CA-4) , CA4- ⁇ Gals, CA-4PD, CA4-NPs and ombrabulin.
  • Taxanes which includes Paclitaxel (Taxol) , a cytotoxic natural product, and docetaxel (Taxotere) , a semi-synthetic derivative, and their analogs which are preferred for conjugation are exampled in: K C. Nicolaou et al., J. Am. Chem. Soc. 117, 2409-20, (1995) ; Ojima et al, J. Med. Chem. 39: 3889-3896 (1996) ; 40: 267-78 (1997) ; 45, 5620-3 (2002) ; Ojima et al., Proc. Natl. Acad. Sci., 96: 4256-61 (1999) ; Kim et al., Bull.
  • Ar and Ar’ are independently aryl or heteroaryl.
  • Anthracyclines are mammalian DNA topoisomerases II inhibitors that are able to stabilize enzyme-DNA complexes wherein DNA strands are cut and covalently linked to the antibody. These anticancer agents maintain a prominent role in treating many forms of solid tumors and acute leukemias during the last several decades.
  • anthracyclines cause cardiovascular morbidity and mortality (Sagi, J.C., et al, Pharmacogenomics. 2016, 17 (9) , 1075-87; McGowan, J.V., et al, Cardiovasc Drugs Ther. 2017, 31 (1) , 63-75) .
  • reasearchers actively are using the conjugation of anthracyclines to a cell-binding antibody, or antibodymolecule as a general approach for improving the therapeutic index of these drugs, (Mollaev, M. et al, Int J Pharm. 2018 Dec 29. pii: S0378-5173 (18) 30991-8; Rossin, R., et al, Bioconjug Chem. 2016, 27 (7) : 1697-706; Dal Corso, A., et al, J Control Release. 2017, 264: 211-218) .
  • anthracyclines include, but are not limited to, daunorubicin, doxorubicin (i.e., adriamycin) , epirubicin, idarubicin, valrubicin, and mitoxantrone.
  • doxorubicin i.e., adriamycin
  • epirubicin i.e., adarubicin
  • valrubicin idarubicin
  • mitoxantrone i.e., mitoxantrone.
  • Vinca alkaloids are a set of anti-mitotic and anti-microtubule alkaloid agents that work by inhibiting the ability of cancer cells to divide.
  • Vinca alkaloids include vinblastine, vincristine, vindesine , leurosine, vinorelbine, catharanthine, vindoline, vincaminol,ieridine, minovincine, methoxyminovincine, minovincinine, vincadifformine, desoxyvincaminol, vincamajine, vincamine, vinpocetine , and vinburnine .
  • the structures of vinca alkaloids are preferred vinblastine, vincristine having the following formula:
  • Dolastatins and their peptidic analogs and derivatives, auristatins are highly potent antimitotic agents that have been shown to have anticancer and antifungal activity. See, e.g., U.S. Pat. No. 5,663,149 and Pettit et al., Antimicrob. Agents Chemother. 42: 2961-2965, 1998.
  • Exemplary dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E (AE) , auristatin EB (AEB) , auristatin EFP (AEFP) , MMAD (Monomethyl Auristatin D or monomethyl dolastatin 10) , MMAF (Monomethyl Auristatin F or N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine) , MMAE (Monomethyl Auristatin E or N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine) , 5-benzoylvaleric acid-AE ester (AEVB) , Auristatin F phenylene diamine (AFP) and other novel auristatins.
  • AE auristatin E
  • AEB auristatin EB
  • AEFP auristatin EFP
  • auristatin analogs are preferred the following formula (Ih-01) , (Ih-02) , (Ih-03) , (Ih-04) , (Ih-05) , (Ih-06) , (Ih-07) , (Ih-08) , (Ih-09) , (Ih-10) , and (Ih-11) :
  • R 1 , R 2 , R 3 , R 4 and R 5 are independently H; C 1 -C 8 linear or branched alkyl, aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, ester, ether, amide, amines, heterocycloalkyl, or acyloxylamines; or peptides containing 1-8 aminoacids, or polyethyleneoxy unit having formula (OCH 2 CH 2 ) p or (OCH 2 CH (CH 3 ) ) p , wherein p is an integer from 1 to about 1000.
  • R 1 R 2 , R 2 R 3 , R 1 R 3 or R 3 R 4 together can form 3 ⁇ 8 member cyclic ring of alkyl, aryl, heteroaryl, heteroalkyl, or alkylcycloalkyl group;
  • Y 1 and Y 2 are independently O, NH, NHNH, NR 5 , S, C (O) O, C (O) NH, OC (O) NH, OC (O) O, NHC (O) NH, NHC (O) S, OC (O) N (R 1 ) , N (R 1 ) C (O) N (R 2 ) , C (O) NHNHC (O) andC (O) NR 1 when linked to the connecting site (that links to L 1 and/or L 2 independently) ; or OH, NH 2 , NHNH 2 , NHR 5 , SH, C (O) OH, C (O) NH 2 , OC (O) NH 2 , OC
  • Hemiasterlin and its analogues bind to the tubulin, disrupt normal microtubule dynamics, and, at stoichiometric amounts, depolymerize microtubules.
  • the structure of maytansinoids is preferred the following formula:
  • R 1 , R 2 , R 3 , R 4 and R 5 are independently H; C 1 -C 8 linear or branched alkyl, aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, ester, ether, amide, amines, heterocycloalkyl, or acyloxylamines; or peptides containing 1-8 aminoacids, or polyethyleneoxy unit having formula (OCH 2 CH 2 ) p or (OCH 2 CH (CH 3 ) ) p , wherein p is an integer from 1 to about 5000;
  • R 2 R 3 can form 3 ⁇ 8 member cyclic ring of alkyl, aryl, heteroaryl, heteroalkyl, or alkylcycloalkyl group.
  • Eribulin which is binding predominantly to a small number of high affinity sites at the plus ends of existing microtubules has both cytotoxic and non-cytotoxic mechanisms of action. Its cytotoxic effects are related to its antimitotic activities, wherein apoptosis of cancer cells is induced following prolonged and irreversible mitotic blockade (Kuznetsov, G. et al, Cancer Research. 2004, 64 (16) : 5760–6.; Towle, M. J, et al, Cancer Research. 2010, 71 (2) : 496–505) .
  • Eribulin has been approved by US FDA for the treatment of metastatic breast cancer who have received at least two prior chemotherapy regimens for late-stage disease, including both anthracycline-and taxane-based chemotherapies, as well as for the treatment of liposarcoma (aspecific type of soft tissue sarcoma) that cannot be removed by surgery (unresectable) or is advanced (metastatic) .
  • Eribulin has been used as payload for ADC conjugates (US20170252458) .
  • the structure of Eribulin is preferred the following formula, Eb01:
  • NAMPT nicotinamide phosphoribosyltransferases
  • NAD + acts as a coenzyme in redox reactions, as a donor of ADP-ribose moieties in ADP-ribosylation reactions, as a precursor of the second messenger molecule cyclic ADP-ribose, as well as acting as a substrate for bacterial DNA ligases and a group of enzymes called sirtuins that use NAD + to remove acetyl groups from proteins.
  • NAD + emerges as an adenine nucleotide that can be released from cells spontaneously and by regulated mechanisms (Smyth L.M, et al, J. Biol. Chem. 2004, 279 (47) , 48893–903; Billington R. A, et al, Mol Med.
  • NAMPT inhibitors are preferred the following formula, NP01, NP02, NP03, NP04, NP05, NP06, NP07, NP08, and NP09:
  • X 5 is F, Cl, Br, I, OH, OR 1 , R 1 , OPO 3 H 2 , OSO 3 H, NHR 1 , OCOR 1 , NHCOR 1 .
  • a benzodiazepine dimer and its analogs are anti-tumor agents that contain one or more immine functional groups, or their equivalents, that bind to duplex DNA.
  • PBD and IGN molecules are based on the natural product athramycin, and interact with DNA in a sequence-selective manner, with a preference for purine-guanine-purine sequences.
  • X 1 , X 2 , Y 1 and Y 2 are independently O, N, NH, NHNH, NR 5 , S, C (O) O, C (O) NH, OC (O) NH, OC (O) O, NHC (O) NH, NHC (O) S, OC (O) N (R 1 ) , N (R 1 ) C (O) N (R 1 ) , CH, C (O) NHNHC (O) andC (O) NR 1 ; R 1 , R 2 , R 3 , R 1’
  • R 1 R 2 , R 2 R 3 , R 1’ R 2’ , or R 2’ R 3’ can independently form 3 ⁇ 8 member cyclic ring of alkyl, aryl, heteroaryl, heteroalkyl, or alkylcycloalkyl group;
  • X 3 and Y 3 are independently N, NH, CH 2 or CR 5, or one ofX 3 and Y 3 can be absent;
  • M 1 and M 2 are independently H, Na, K, Ca, Mg, NH 4 , NR 1 R 2 R 3 ;
  • X 6 is CH, N, P (O) NH, P (O) NR 1 , CHC (O) NH, C 3 -C 8 aryl, heteroaryl, alkylcycloalkyl, acyloxyl, alkylaryl, alkylaryloxyl, alkylarylamino, or an Aa (amino acid, is preferably selected from Lys, Phe, Asp, Glu, Ser, Thr, His, Cys, Tyr, Trp, Gln, Asn, Arg) ; is defined the same above.
  • CC-1065 analog and doucarmycin analogs are also preferred to be used for a conjugate of the present process invention.
  • the examples of the CC-1065 analogues and doucarmycin analogs as well as their synthesis are described in: e.g. Warpehoski, et al, J. Med. Chem. 31: 590-603 (1988) ; D. Boger et al., J. Org. Chem; 66; 6654-61, 2001; U.S.
  • X 1 , X 2 , Y 1 and Y 2 are independently O, NH, NHNH, NR 5 , S, C (O) O, C (O) NH, OC (O) NH, OC (O) O, NHC (O) NH, NHC (O) S, OC (O) N (R 1 ) , N (R 1 ) C (O) N (R 2 ) , C (O) NHNHC (O) andC (O) NR 1 when linked to the connecting site or OH, NH 2 , NHNH 2 , NHR 1 , SH, C (O) OH, C (O) NH 2 , OC (O) NH 2 , OC (O) OH, NHC (O) NH 2 , NHC (O) SH, OC (O) NH (R 1 ) , N (R 1 ) C (O) NH (O) NH (R 2 ) , C (O) NHNHC (
  • amatoxin and its analogs which are a subgroup of at least ten toxic compounds originally found in several genera of poisonous mushrooms, most notably Amanita phalloides and several other mushroom species, are also preferred for conjugation of the present patent.
  • These ten amatoxins named ⁇ -Amanitin, ⁇ -Amanitin, ⁇ -Amanitin, ⁇ -Amanitin, Amanullin, Amanullinic acid, Amaninamide, Amanin, Proamanullin, are rigid bicyclic peptides that are synthesized as 35-amino-acid proproteins, from which the final eight amino acids are cleaved by a prolyl oligopeptidase (Litten, W.
  • Spliceostatins and pladienolides are anti-tumor compounds which inhibit splicing and interacts with spliceosome, SF3b.
  • spliceostatins include, but are not limited to, spliceostatin A, FR901464, and (2S, 3Z) -5- ⁇ [ (2R, 3R, 5S, 6S) -6- ⁇ (2E, 4E) -5- [ (3R, 4R, 5R, 7S) -7- (2-hydrazinyl-2-oxoethyl) -4-hydroxy-1, 6-dioxaspiro [2.5] oct-5-yl] -3-methylpenta-2, 4-dien-1-y-l ⁇ -2, 5-dimethyltetrahydro-2H-pyran-3-yl] amino ⁇ -5-oxopent-3-en-2-yl acetate having the core structure:
  • pladienolides examples include, but are not limited to, Pladienolide B, Pladienolide D, and E7107.
  • Protein kinase inhibitors that block the action of an enzyme to add a phosphate (PO 4 ) group to serine, threonine, or tyrosine amino acids on an antibody, and can modulate the protein function.
  • the protein kinase inhibitors can be used to treat diseases due to hyperactive protein kinases (including mutant or overexpressed kinases) in cancer or to modulate cell functions to overcome other disease drivers.
  • protein kinase inhibitors are preferred to selected from Adavosertib, Afatinib, Axitinib, Bafetinib, Bosutinib, Cobimetinib, Crizotinib, Cabozantinib, Dasatinib, Entrectinib, Erdafitinib, Erlotinib, Erlotinib, Fostamatinib, Gefitinib, Ibrutinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Pazopanib, Pegaptanib, Ponatinib, Rebastinib, Regorafenib, Ruxolitinib, Sorafenib, Sunitinib, SU6656, Tofacitinib, Vandetanib, Vemurafenib, Entrectinib, Palbociclib, Ribociclib, Riboc
  • Z 5 and Z 5 ’ are independently selected from O, NH, NHNH, NR 5 , S, C (O) O, C (O) NH, OC (O) NH, OC (O) O, NHC (O) O, NHC (O) NH, NHC (O) S, OC (O) N (R 1 ) , N (R 1 ) C (O) N (R 2 ) , C (O) NHNHC (O) andC (O) NR 1 .
  • a MEK inhibitor inhibits the mitogen-activated protein kinases MEK1 and/or MEK2 which is often overactive in some cancers.
  • MEK inhibitors are especially used for treatment of BRAF-mutated melanoma, and KRAS/BRAF mutated colorectal cancer, breast cancer, and non-small cell lung cancer (NSCLC) .
  • MEK inhibitors are selected from PD0325901, selumetinib (AZD6244) , cobimetinib (XL518) , refametinib, trametinib (GSK1120212) , pimasertib, Binimetinib (MEK162) , AZD8330, RO4987655, RO5126766, WX-554, E6201, GDC-0623, PD-325901 and TAK-733.
  • the preferred MEK inhibitors are selected from Trametinib (GSK1120212) , Cobimetinib (XL518) , Binimetinib (MEK162) , selumetinib having the following formula:
  • Z 5 is selected from O, NH, NHNH, NR 5 , S, C (O) O, C (O) NH, OC (O) NH, OC (O) O, NHC (O) O, NHC (O) NH, NHC (O) S, OC (O) N (R 1 ) , N (R 1 ) C (O) N (R 2 ) , C (O) NHNHC (O) andC (O) NR 1 ;
  • a proteinase inhibitor that are used as a payload is preferably selected from: Carfilzomib, Clindamycin, Rumblemulin, Indibulin, as shown in the following structures:
  • An immunotoxin herein is a macromolecular drug which is usually a cytotoxic protein derived from a bacterial or plant protein, such as Diphtheria toxin (DT) , Cholera toxin (CT) , Trichosanthin (TCS) , Dianthin, Pseudomonas exotoxin A (ETA′) , Erythrogenic toxins, Diphtheria toxin, AB toxins, Type III exotoxins, etc. It also can be a highly toxic bacterial pore-forming protoxin that requires proteolytic processing for activation. An example of this protoxin is proaerolysin and its genetically modified form, topsalysin.
  • Topsalysin is a modified recombinant protein that has been engineered to be selectively activated by an enzyme in the prostate, leading to localized cell death and tissue disruption without damaging neighboring tissue and nerves;
  • An immunotoxin herein is preferably conjugated via the process of the application through an amino acid having free amino, thiol or carboxyl acid group; and more preferably through N-terminal amino acid.
  • a certain cell receptor agonist, a cell stimulating molecule or intracellular signallingmolecule can be as a chemotherapeutic /function compound conjugated to BCMA antibody of the invention.
  • Acell-binding ligand or receptor agonist selected from: Folate derivatives; Glutamic acid urea derivatives; Somatostatin and its analogs (selected from the group consisting of octreotide (Sandostatin) and lanreotide (Somatuline) ) ; Aromatic sulfonamides; Pituitary adenylate cyclase activating peptides (PACAP) (PAC1) ; Vasoactive intestinal peptides (VIP/PACAP) (VPAC1, VPAC2) ; Melanocyte-stimulating hormones ( ⁇ -MSH) ; Cholecystokinins (CCK) /gastrin receptor agonists; Bombesins (selected from the group consisting ofPyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH 2 ) /gastrin-releasing peptide (
  • Acell-binding molecule/ligand or a cell receptor agonists selected from the following: LB01 (Folate) , LB02 (PMSA ligand) , LB03 (PMSA ligand) , LB04 (PMSA ligand) , LB05 (Somatostatin) , LB06 (Somatostatin) , LB07 (Octreotide, a Somatostatin analog) , LB08 (Lanreotide, a Somatostatin analog) , LB09 (Vapreotide (Sanvar) , a Somatostatin analog) , LB10 (CAIX ligand) , LB11 (CAIX ligand) , LB12 (Gastrin releasing peptide receptor (GRPr) , MBA) , LB13 (luteinizing hormone-releasing hormone (LH-RH) ligand and GnRH) , LB14 (luteinizing hormone-releasing hormone (LH-
  • X 4 , and Y 1 are independently O, NH, NHNH, NR 1 , S, C (O) O, C (O) NH, OC (O) NH, OC (O) O, NHC (O) NH, NHC (O) S, OC (O) N (R 1 ) , N (R 1 ) C (O) N (R 1 ) , CH 2 , C (O) NHNHC (O) andC (O) NR 1 .
  • one, two or more DNA, RNA, mRNA, small interfering RNA (siRNA) , microRNA (miRNA) , and PIWI interacting RNAs (piRNA) can be as a chemotherapeutic /function compound conjugated to BCMA antibody of the invention:
  • X 1 , and Y are independently O, NH, NHNH, NR 1 , S, C (O) O, C (O) NH, OC (O) NH, OC (O) O, NHC (O) NH, NHC (O) S, OC (O) N (R 1 ) , N (R 1 ) C (O) N (R 1 ) , CH 2 , C (O) NHNHC (O) andC (O) NR 1 .
  • the linker L 1 and L 2 are, the same or different, independently selected from O, NH, S, S-S, NHNH, N (R 3 ) , N (R 3 ) N (R 3’ ) , C 1 -C 8 of alkyl; C 2 -C 8 of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C 3 -C 8 of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; C 2 -C 8 (2-8 carbon atoms) of esters, ether, or amide; 1 ⁇ 8 natural or unnatural amino acids described in the definition; polyethyleneoxy unit of formula (OCH 2 CH 2 ) p , (OCH 2 CH (CH 3 ) ) p , (OCH 2 CH 2 ) p OR 3 , (OCH 2 CH (CH 3 ) p OR 3 , NH
  • L 1 or L 2 may contain a self-immolative or a non-self-immolativecomponent, peptidyl units, a hydrazone bond, a disulfide, an ester, an oxime, an amide, or a thioether bond.
  • the self-immolativeunit includes, but is not limited to, aromatic compounds that are electronically similar to the para-aminobenzylcarbamoyl (PAB) groups such as 2-aminoimidazol-5-methanol derivatives, heterocyclic PAB analogs, beta-glucuronide, and ortho or para-aminobenzylacetals.
  • PAB para-aminobenzylcarbamoyl
  • the self-immolativelinker component has one of the following structures:
  • X 1 , Y 1 , Z 2 and Z 3 are independently NH, O, or S;
  • Z 1 is independently H, NH, O or S;
  • v is 0 or 1;
  • the non-self-immolativelinker component is one of the following structures:
  • the (*) atom is the point of attachment of additional spacer R 1 or releasable linkers, the cytotoxic agents, and/or the binding molecules;
  • X 1 , Y 1 , U 1 , R 1 , R 5 , R 5 ’ are defined as above;
  • r is 0 ⁇ 100;
  • m and n are 0 ⁇ 6 independently.
  • L 1 or L 2 may be composed of one or more linker components of 6-maleimidocaproyl ( “MC” ) , maleimidopropanoyl ( “MP” ) , valine-citrulline ( “val-cit” or “vc” ) , alanine-phenylalanine ( “ala-phe” or “af” ) , p-aminobenzyloxycarbonyl ( “PAB” ) , 4-thiopentanoate ( “SPP” ) , 4- (N-maleimidomethyl) cyclohexane-1 carboxylate ( “MCC” ) , (4-acetyl) amino-benzoate ( “SIAB” ) , 4-thio-butyrate (SPDB) , 4-thio-2-hydroxysulfonyl-butyrate (2-Sulfo-SPDB) , or natural or unnatural peptides having 1 ⁇ 8 natural or unnatural amino acid unites.
  • L 1 or L 2 may be a releasable linker.
  • the term releasable linker refers to a linker that includes at least one bond that can be broken under physiological conditions, such as a pH-labile, acid-labile, base-labile, oxidatively labile, metabolically labile, biochemically labile, or enzyme-labile bond.
  • physiological conditions resulting in bond breaking do not necessarily include a biological or metabolic process, and instead may include a standard chemical reaction, such as a hydrolysis or substitution reaction, for example, an endosome having a lower pH than cytosolic pH, and/or disulfide bond exchange reaction with a intracellular thiol, such as a millimolar range of abundant of glutathione inside the malignant cells.
  • a standard chemical reaction such as a hydrolysis or substitution reaction, for example, an endosome having a lower pH than cytosolic pH, and/or disulfide bond exchange reaction with a intracellular thiol, such as a millimolar range of abundant of glutathione inside the malignant cells.
  • releasable linkers examples include, but not limited:
  • Example structures of the components of the linker L 1 and L 2 may contain:
  • X 2 , X 3 , X 4 , X 5 , orX 6 are independently selected from NH; NHNH; N (R 12 ) ; N (R 12 ) N (R 12’ ) ; O; S; C 1 -C 6 of alkyl; C 2 -C 6 of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C 3 -C 8 of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; CH 2 OR 12 , CH 2 SR 12 , CH 2 NHR 12 , or 1 ⁇ 8 amino acids; wherein R 12 and R 12’ are independently H; C 1 -C 8 of alkyl; C 2 -C 8 of hetero-alkyl, alkylcycloalkyl, heterocycloalkyl; C 3 -
  • the L 1 and L 2 are independentlyselectedfrom:
  • ⁇ is a site that links a drug or a site of linker L 1 or L 2 ;
  • Aa is L-or D-natural or unnatural amino acids;
  • r is 0-12; when r isnot 0, (Aa) r isthesameordifferentamino acids or peptide units;
  • the L 1 and L 2 are independentlyselectedfrom:
  • the conjugates of Formula (I) , (II) and (III) are prepared via conjugation reaction of the antibody with compounds having the following formula (IV) , (V) and (VI) respectively:
  • Lv 1 and Lv 2 are a reactive group, and are independently selected from:
  • X 1 ’ and X 2 ’ are independently F, Cl, Br, I, OTf, OMs, OC 6 H 4 (NO 2 ) , OC 6 H 3 (NO 2 ) 2 , OC 6 F 5 , OC 6 HF 4 , or Lv 3 ;
  • X 2 is O, NH, N (R 1 ) , or CH 2 ;
  • R 3 and R 5 are independently H, R 1 , aromatic, heteroaromatic, or aromatic group wherein one or several H atoms are replaced independently by -R 1 , -halogen, -OR 1 , -SR 1 , -NR 1 R 2 , -NO 2 , -S (O) R 1 , -S (O) 2 R 1 , or -COOR 1 ;
  • Lv 3 and Lv 3 ’ are independently a leaving group selected from F, Cl, Br, I, nitrophenol; N-hydroxysuccinimide (NHS) ; phenol; benzen
  • L 1 , L 2 , E 1 , Lv 1 , and Lv 2 are defined the same above for Formula (I) , (II) (III) . (IV) , (V) , and (VI) ; wherein Lv 5 and Lv 6 are independently selected from
  • X 1 ’ is F, Cl, Br, I, OTs (tosylate) , OTf (triflate) , OMs (mesylate) , OC 6 H 4 (NO 2 ) , OC 6 H 3 (NO 2 ) 2 , OC 6 F 5 , OC 6 HF 4 , or Lv 3 ;
  • X 2 ’ is O, NH, N (R 1 ) , or CH 2 ;
  • R 3 and R 5 are independently H, R 1 , aromatic, heteroaromatic, or aromatic group wherein one or several H atoms are replaced independently by -R 1 , -halogen, -OR 1 , -SR 1 , -NR 1 R 2 , -NO 2 , -S (O) R 1 , -S (O) 2 R 1 , or -COOR 1 ;
  • Lv 3 and Lv 3 ’ are independently a leaving group selected from F, Cl, Br, I, nitrophenoxyl;
  • the conjugates of the present patent invention can be made through introducation of a certain fuction group in the antibody, typically generation of thiols between heavy-light chain when the antibody is IgG antibody, then the thiols simultaneously or sequentially in the conjugation process react to the linker of formula (VII) , (VIII) or (IX) illustrated above to form the antibody/linker complex molecule of formula (X) , (XI) or (XII) below, following by reaction with a a cytotoxic drug D 1 or D 2 independently to form the conjugate of formula (I) , (II) , or (III) .
  • linker of formula (VII) , (VIII) or (IX) illustrated above can react first with a cytotoxic drug to form the cytotoxic drug/linker complex molecule of formula (IV) , (V) or (VI) , follow by reaction with the reduced a fuction group in the antibody independently to form the conjugate of formula (I) , (II) , or (III) .
  • the first step condensation reaction of the formula (VII) , (VIII) or (IX) to a cytotoxic drug can be in a separated pot, and the resulted cytotoxic drug/linker complex molecules of formula (IV) , (V) or (VI) can beoptionally purified by a chromatography, extraction or precipitatation before for conjugation to the fuction group in the antibody.
  • the conjugation reaction with formula (IV) , (V) or (VI) is preferred in the same pot without separation ofintermidiates.
  • each step of the reactions for the linker of formula (VIII) , (IX) or (X) can be conducted at different conditions in the same or different reaction pots.
  • a drug containing an amino group can undergo condensation with a carboxylic acid group in the linker in the present of a condensation regent, e.g. EDC, TBTU or BroP, to give a modified drug/linker complex of Formula (IV) , (V) or VI) bearing amide bonds.
  • This condensation reaction can be performed at physiological buffer solution wherein the carboxylic acid group at one terminal of the linker of formula (VII) , (VIII) or (IX) is activated to be N-hydoxylsuccinimidyl (NHS) , pentfluorophenyl, dinitrophenyl ester, or carboxylic acid chloride group, etc, which can react to a drug bearing an amino group to provide drug/linker complex of Formula (III) , (IV) or V) , then subsequently or simultaneously undergo the conjugation to thiols of the antibody to form the conjugate of formula (I) , (II) , or (III) .
  • NHS N-hydoxylsuccinimidyl
  • pentfluorophenyl pentfluorophenyl, dinitrophenyl ester, or carboxylic acid chloride group, etc, which can react to a drug bearing an amino group to provide drug/linker complex of Formula (III) , (IV
  • linker of formula (VII) , (VIII) or (IX) bearing both a thiol reactive group (e.g. maleimido, vinylsulfonyl, haloacetyl, acrylic, substitutedpropiolic) at one terminal and a drug reactive group (e.g. hydoxylsuccinimidyl (NHS) , pentfluorophenyl, dinitrophenyl ester, amino, alkyloxylamino or clickable chemistry group (e.g.
  • a thiol reactive group e.g. maleimido, vinylsulfonyl, haloacetyl, acrylic, substitutedpropiolic
  • a drug reactive group e.g. hydoxylsuccinimidyl (NHS)
  • pentfluorophenyl, dinitrophenyl ester amino, alkyloxylamino or clickable chemistry group
  • the antibody--linker conjugate of formula (X) , (XI) or (XII) can be optionally purified before proceeding the condensation with a drug, and the condensation condition of the second step can be adjusted, e.g. the pH can be adjusted to 6.5 –8.0, and/or temperature can be adjusted to 20 -45 °C if needed.
  • the antibody can be modified through attachment of a heterobifunctional cross linker of formula (X) , (XI) or (XII) , such as with linkers of Amine-to-Sulfhydryl (succinimidyl (NHS) ester/maleimide, NHS ester/pyridyldithiol, NHS esters/haloacetyl) , diazirine (SDA) –to-Sulfhydryl, Azide-to-Sulfhydryl, Alkyne-to-Sulfhydryl, Sulfhydryl-to-Carbohydrate (Maleimide/Hydrazide, Pyridyldithiol /Hydrazide, haloacetyl /Hydrazide) , Hydroxyl-to-Sulfhydryl (Isocyanate/Maleimide)
  • the reactive group of a drug/cytotoxic agent that reacting to a modified antibody-linker conjugate of formula (X) , (XI) or (XII) to give the final conjugate can be in different ways accordingly.
  • the conjugate linked via disulfide bonds is achieved via the first step, a linker of formula (VII) , (VIII) or (IX) is conjugated to the antibody at 2 °C -8 °C, pH 4.5 –6.0, following by a disulfideexchange between a drug containing a free thiol group and the disulfide bond (e.g.
  • the excess reduction agent e.g. TCEP, or tri (3-hydroxylpropyl) phosphine
  • an azide compound e.g. 4- (azidomethyl) benzoic acid
  • Synthesis of the conjugates linked via thioether is achieved by first reaction of a linker containing both thiol reactive terminals of maleimido or haloacetyl or ethylsulfonyl or substitutedpropiolic group to the thiols in the antibody which are reduced by the process of the present patent application at 2 °C -8 °C, pH 4.5 –6.5 to give the antibody-linker conjugate of formula (X) , (XI) or (XII) , following by reaction of a drug containing a thiol at pH 6.5 –8.0, at 20 °C -40 °C to to provide the conjugate of formula (I) , (II) , or (III) .
  • the preferred methods of synthesis of the disulfide or thiol-ether linked conjugates are through the first chemical synthesis the drug-linker complex having disulfide or thiol-ether bonds of the formula (IV) , (V) or (VI) ; following by reaction with the thiols in the protein (antibody) according the process of the invention.
  • Synthesis of conjugates bearing an acid labile hydrazone linkage can be achieved by reaction of a carbonyl group with the hydrazide moiety in the linker, by methods known in the art (see, for example, P. Hamann et al., Cancer Res. 53, 3336-34, 1993; B. Laguzza et al., J. Med. Chem., 32; 548-55, 1959; P. Trail et al., Cancer Res., 57; 100-5, 1997) .
  • Synthesis of conjugates bearing triazole linkage can be achieved by reaction of a 1-yne group of the drug with the azido moiety in the linker, through the click chemistry (Huisgen cycloaddition) (Lutz, J-F.
  • Synthesis of the conjugates linked via oxime is achieved by reaction of a modified antibody containing a ketone or aldehyde and a drug containing oxyamine group.
  • a drug bearing a hydroxyl group or a thiol group can be reacted with a modified linker of Formula (X) , (XI) , or (XII) , bearing a halogen, particularly the alpha halide of carboxylates, in the presence of a mild base, e.g.
  • a drug containing a hydroxyl group can be condensed with a linker of Formula (X) , (XI) , or (XII) bearing a carboxyl group, in the presence of a dehydrating agent, such as EDC or DCC, to give ester linkage, then the subject drug/linker complex undergoes the conjugation with an antibody under the process of the present invention.
  • a dehydrating agent such as EDC or DCC
  • a drug containing an amino group can condensate with a carboxyl ester of NHS, imidazole, nitrophenoxyl; N-hydroxysuccinimide (NHS) ; methylsufonylphenoxyl; dinitrophenoxyl; pentafluorophenoxyl; tetrafluorophenoxyl; difluorophenoxyl; monofluorophenoxyl; pentachlorophenoxyl; triflate; imidazole; dichlorophenoxyl; tetrachlorophenoxyl; 1-hydroxyben-zotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3′-sulfonate in the antibody-linker of Formula (X) , (XI) or (XII) to give a conjugate via amide bond linkage of Formula (I) , (II) , or (III) .
  • NHS N-hydroxysuccinimide
  • the BCMA antibody conjugates are preferably prepared via ahomogenous conjugationprocess, which comprisesthefollowing three keysteps:
  • PBS Mes, Bis-Tris, Bis-Tris Propane, Pipes, Aces, Mopso, Bes, Mops, Hepes, Tes, Pipps, Dipso, Tapso, Heppso, Tris-up, Tris-HCl, Tricine, Hepps, Gly-Gly, Bicine, Taps, Hepee, Acetates, Histidine, Citrates, MES, or Borates, etc. ) toselectively reduceinterchaindisulfidebondswithintheantibody, to generate thiols;
  • the step (c) can be replaced by: adding an effective amount of cystine to quench the excessive conjugation linker or linker/payload complex containing thiol reactive groups (e.g. maleimide) ; and simultaneously or sequentially addingan azido compound (e.g. 4- (azidomethyl) -benzoic acid) ora disulfide compound (e.g. cystine) to quench the unreacted reductant (e.g. TCEP or Tris (hydroxypropyl) phosphine) .
  • the addition of cystine to to quench the unreacted reductant (e.g. TCEP) can form a cysteine which cansimultaneouslyquench the excessive conjugation linker or linker/payload complex containing thiol reactive groups (e.g. maleimide) .
  • R 1 , R 2 and R 3 in the formula of Zn (NR 1 R 2 R 3 ) m1 2+ are independently selected from C 1 -C 8 of alkyl; C 2 -C 8 of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C 3 -C 8 of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; m1 is selected from 1, 2, 3, 4, 5, 6, 7 or 8; Proferably m1 is 1, 2, 3 or 4.
  • (NR 1 R 2 R 3 ) m1 can be form a dimer, trimer, tetramer, pentamer, or hexamer wherein these polymers are covalently linked among N, R 1 , R 2 and R 3 ; and N, R 1 , R 2 or R 3 themselveor together can form heterocyclic, carbocyclic, diheterocyclic, or dicarbocyclic rings.
  • TheZinc cation-amino chelate/complex, Zn (NR 1 R 2 R 3 ) m1 2+ , used in step (a) is 0.01mM–1.0mM in concentration, or 0.5 ⁇ 20 equivalents in moles of the protein, and it can be added to the reaction solution with a water-soluble organic solvent, selected from, ethanol, methanol, propanol, propandiol, DMA, DMF, DMSO, THF, CH 3 CN.
  • a water-soluble organic solvent selected from, ethanol, methanol, propanol, propandiol, DMA, DMF, DMSO, THF, CH 3 CN.
  • an organic phosphine preferably selected fromTris (2-carboxyethyl) -phosphine (TECP) or Tris (hydroxypropyl) phosphine and itsuseinthereactionsolutionis0.02mM–1.0mM in concentration, or 1.0 –20 equivalents in moles of the protein.
  • Theoxidanttobeaddedinstep (c) maybeDHAA, Fe 3+ , I 2 , Cu 2+ , Mn 3+ , MnO 2 , or mixture of Fe 3+ /I - .
  • the oxidant used inthereactionsolution is0.02mM-1.0mM in concentration, or 0.2 -100 equivalents in moles of the protein.
  • Theoptimal reaction conditions e.g.
  • pH, temeperature, buffer, concentrations of the reactants of course are dependeduponspecifically an antibody-like protein, a payload/linker complex, areductant and/or Zn (NR 1 R 2 R 3 ) m1 2+ used.
  • the resulted conjugates of formula (I) , (II) , or (III) are over 75%linked to the cysteine sites between heavy-light chains of an antibody, and are less than 15%linked to the cysteine sites between heavy-heavy chains (hinge region) of an antibody.
  • DAR drug/antibody ratio
  • the distributions in percentage of the numbers of drugs in the antibody are: D0 ⁇ 1%, D2 ⁇ 10%, D4>65%, D6 ⁇ 10%, D8 ⁇ 10%; for formula (III) .
  • the resulted conjugate may be purified by standard biochemical means, such as gel filtration on a Sephadex G25 or Sephacryl S300 column, adsorption chromatography, ion (cation or anion) exchange chromatography, affinity chromatography (e.g. protein A column) or by dialysis (ultrafiltration or hyperfiltration (UF) and diafiltration (DF) ) .
  • a small size molecule of antibody e.g. ⁇ 100 KD
  • conjugated with a small molecular drugs can be purified by chromatography such as by HPLC, medium pressure column chromatography or ion exchange chromatography.
  • the conjugate of Formula (I) , (II) , or (III) is preferably generated from a drug/linker complex of Formula (IV) , (V) , or (VI) , as in a one pot reaction.
  • the Ellman reagent can be optionally used to monitor the efficient reduction of the disulfide bonds and conjugation of the tiols through measurement of the numbers of the free thiols during the reactions.
  • a UV spectrometry at wavelength of range 190-390 nm, preferably at 240-380 nm, more preferably at 240-370 nm is preferred to be used in assisting the reaction (via monitoring the conjugation) .
  • the conjugation reaction can be thus measured or conducted in a quartz cell or Pyrex flask in temperature control environment.
  • the drug/protein (antibody) ratios (DAR) of the conjugates can also be measured by UV at wavelength of range 240-380 nmvia calculation of the concentrations of the drug and the protein, by Hydrophobic Interaction Chromatography (HIC-HPLC) via measurement of the integration areas of each drug/protein fragment, by Capilaryelectrophoresis (CE) , and/or by LC-MS or LC-MS/MS or CE-MS (the combination ofliquid chromatography (LC) or CE withmass spectrometry (MS) via measurement of both the integration areas of LC or CE and Peak intensity of MS for each drug/protein fragment) .
  • HIC-HPLC Hydrophobic Interaction Chromatography
  • CE Capilaryelectrophoresis
  • CE-MS the combination ofliquid chromatography (LC) or CE withmass spectrometry (MS) via measurement of both the integration areas of LC or CE and Peak intensity of MS for each drug/protein fragment
  • a drug or a drug/linker complex when a drug or a drug/linker complex is not well soluble in a water based buffer solution, up to 30%of water mixable (miscible) organic solvents, such as DMA, DMF, ethanol, methanol, acetone, acetonitrile, THF, isopropanol, dioxane, propylene glycol, or ethylene diol can be added as the co-solvent in water based buffer solution.
  • water mixable organic solvents such as DMA, DMF, ethanol, methanol, acetone, acetonitrile, THF, isopropanol, dioxane, propylene glycol, or ethylene diol
  • the aqueous solutions for the modification of the antibody are buffered between pH 4 and 9, preferably between 6.0 and 7.5 and can contain any non-nucleophilic buffer salts useful for these pH ranges.
  • Typical buffers include phosphate, acetate, triethanolamine HCl, HEPES, and MOPS buffers, which can contain additional components, such as cyclodextrins, sucrose and salts, for examples, NaCl and KCl.
  • Other biological buffers that are used for the conjugation process are listed in the definition section.
  • the progress of the reaction can be monitored by measuring the decrease in the absorption at a certain UV wavelength, such as at 254 nm, or increase in the absorption at a certain UV wavelength, such as 280 nm, or the other appropriate wavelength.
  • isolation of the modified cell-binding antibody agent can be performed in a routine way, using for example gel filtration chromatography, or adsorptive chromatography.
  • the extent of the modification can be assessed by measuring the absorbance of the nitropyridine thione, dinitropyridinedithione, pyridine thione, carboxylamidopyridinedithione and dicarboxyl-amidopyridinedithione group released via UV spectra.
  • the modification or conjugation reaction can be monitored by LC-MS, preferably by UPLC-QTOF mass spectrometry, or Capilaryelectrophoresis–mass spectrometry (CE-MS) .
  • the linker compounds have diverse functional groups that can react with drugs, preferably cytotoxic agents that possess a suitable substituent.
  • the modified antibody bearing an amino or hydroxyl substituent can react with drugs bearing an N-hydroxysuccinimide (NHS) ester
  • the modified antibody bearing a thiol substituent can react with drugs bearing a maleimido or haloacetyl group
  • the modified antibody bearing a carbonyl (ketone or aldehyde) substituent can react with drugs bearing a hydrazide or an alkoxyamine.
  • the BCMA antibody conjugates of the patent application are formulated to liquid, or suitable to be lyophilized and subsequently be reconstituted to a liquid formulation.
  • the conjugate in a liquid formula or in the formulated lyophilized powder may take up 0.01%-99%by weight as major gradient in the formulation.
  • a liquid formulationcomprising 0.1 g/L ⁇ 300 g/L of concentration of the conjugate active ingredient for delivery to a patient without high levels of antibody aggregation may include one or more polyols (e.g. sugars) , a buffering agent with pH 4.5 to 7.5, a surfactant (e.g. polysorbate 20 or 80) , an antioxidant (e.g.
  • a tonicity agent e.g. mannitol, sorbitol or NaCl
  • chelating agents such as EDTA
  • metal complexes e.g. Zn-protein complexes
  • biodegradable polymers such as polyesters
  • a preservative e.g. benzyl alcohol
  • Suitable buffering agents for use in the formulations include, but are not limited to, organic acid salts such as sodium, potassium, ammounium, or trihydroxyethylaminosalts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phtalic acid; Tris, tromethamine hydrochloride, sulfate or phosphate buffer.
  • amino acid cationic components can also be used as buffering agent.
  • amino acid component includes without limitation arginine, glycine, glycylglycine, and histidine.
  • the arginine buffers include arginine acetate, arginine chloride, arginine phosphate, arginine sulfate, arginine succinate, etc.
  • the arginine buffer is arginine acetate.
  • histidine buffers include histidine chloride-arginine chloride, histidine acetate-arginine acetate, histidine phosphate-arginine phosphate, histidine sulfate-arginine sulfate, histidine succinate-argine succinate, etc.
  • the formulations of the buffers have a pH of 4.5 to pH 7.5, preferably from about 4.5 to about 6.5, more preferably from about 5.0 to about 6.2.
  • the concentration of the organic acid salts in the buffer is from about 10 mM to about 500 mM.
  • a “polyol” that may optionally be included in the formulation is a substance with multiple hydroxyl groups.
  • Polyols can be used as stabilizing excipients and/or isotonicity agents in both liquid and lyophilized formulations.
  • Polyols can protect biopharmaceuticals from both physical and chemical degradation pathways.
  • Preferentially excluded co-solvents increase the effective surface tension of solvent at the protein interface whereby the most energetically favorable structural conformations are those with the smallest surface areas.
  • Polyols include sugars (reducing and nonreducing sugars) , sugar alcohols and sugar acids.
  • a “reducing sugar” is one which contains a hemiacetal group that can reduce metal ions or react covalently with lysine and other amino groups in proteins and a “nonreducing sugar” is one which does not have these properties of a reducing sugar.
  • reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose.
  • Nonreducing sugars include sucrose, trehalose, sorbose, melezitose and raffinose.
  • Sugar alcohols are selected from mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, threitol, sorbitol and glycerol.
  • Sugar acids include L-gluconate and metallic salts thereof.
  • the polyol in the liquid formula or in the formulated lyophilized solid can be 0.0%-20%by weight.
  • a nonreducing sugar, sucrose or trehalose at a concentration of about from 0.1%to 15% is chosen in the formulation, wherein trehalose being preferred over sucrose, because of the solution stability of trehalose.
  • a surfactant optionally in the formulations is selected from polysorbate (polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85 and the like) ; poloxamer (e.g. poloxamer 188, poly (ethylene oxide) -poly (propylene oxide) , poloxamer 407 or polyethylene-polypropylene glycol and the like) ; Triton; sodium dodecyl sulfate (SDS) ; sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropy
  • lauroamidopropyl myristamidopropyl-, palmidopropyl-, or isostearamido-propyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; dodecyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine and coco ampho glycinate; and the MONAQUAT TM series (e.g. isostearylethylimidoniumethosulfate) ; polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc) ; etc.
  • Pluronics, PF68 etc ethylene and propylene glycol
  • Preferred surfactants are polyoxyethylenesorbitan fatty acid esters e.g. polysorbate 20, 40, 60 or 80 (Tween 20, 40, 60 or 80) .
  • the concentration of a surfactant in the formulation is range from 0.0%to about 2.0%by weight. In certain embodiments, the surfactant concentration is from about 0.01%to about 0.2%. In one embodiment, the surfactant concentration is about 0.02%.
  • a “preservative” optionally in the formulations is a compound that essentially reduces bacterial action therein.
  • potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds) , and benzethonium chloride.
  • preservatives include aromatic alcohols such as phenoxyl, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.
  • aromatic alcohols such as phenoxyl, butyl and benzyl alcohol
  • alkyl parabens such as methyl or propyl paraben
  • catechol resorcinol
  • cyclohexanol 3-pentanol
  • m-cresol m-cresol
  • the preservative in the liquid formula or in the formulated lyophilized powder can be 0.0%-5.0%by weight.
  • the preservative herein is benzyl alcohol.
  • Suitable free amino acids as a bulky material, or tonicity agent, or osmotic pressure adjustment in the formulation is selected from, but are not limited to, one or more of arginine, cystine, glycine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine glutamic acid or aspartic acid.
  • arginine, cystine, glycine, lysine, histidine, ornithine isoleucine, leucine, alanine, glycine glutamic acid or aspartic acid.
  • the inclusion of a basic amino acid is preferred i.e. arginine, lysine and/or histidine. If a composition includes histidine then this may act both as a buffering agent and a free amino acid, but when a histidine buffer is used it is typical to include a non-histidine free amino acid e.g.
  • amino acid may be present in its D-and/or L-form, but the L-form is typical.
  • the amino acid may be present as any suitable salt e.g. a hydrochloride salt, such as arginine-HCl.
  • the amino acid in the liquid formula or in the formulated lyophilized powder can be 0.0%-30%by weight.
  • the formulations can optionally comprise methionine, glutathione, cysteine, cystine or ascorbic acid as an antioxidant at a concentration of about up to 5 mg/ml in the liquid formula or 0.0%-5.0%by weight in the formulated lyophilized powder;
  • the formulations can optionally comprise metal chelating agent, e.g., EDTA, EGTA, etc., at a concentration of about up to 2 mM in the liquid formula or 0.0%-0.3%by weight in the formulated lyophilized powder.
  • the final formulation can be adjusted to the preferred pH with a buffer adjusting agent (e.g. an acid, such as HCl, H 2 SO 4 , acetic acid, H 3 PO 4 , citric acid, etc, or a base, such as NaOH, KOH, NH 4 OH, ethanolamine, diethanolamine or triethanol amine, sodium phosphate, potassium phosphate, trisodium citrate, tromethamine, etc) and the formulation should be controlled “isotonic” which is meant that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsm.
  • a buffer adjusting agent e.g. an acid, such as HCl, H 2 SO 4 , acetic acid, H 3 PO 4 , citric acid, etc, or a base, such as NaOH, KOH, NH 4 OH, ethanolamine, diethanolamine or triethanol amine, sodium phosphat
  • Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example.
  • the isotonic agent is selected from mannitol, sorbitol, sodium acetate, potassium chloride, sodium phosphate, potassium phosphate, trisodium citrate, or NaCl.
  • both the buffer salts and the isotonic agent may take up to 30%by weight in the formulation.
  • excipients which may be useful in either a liquid or lyophilized formulation of the patent application include, for example, fucose, cellobiose, maltotriose, melibiose, octulose, ribose, xylitol, arginine, histidine, glycine, alanine, methionine, glutamic acid, lysine, imidazole, glycylglycine, mannosylglycerate, Triton X-100, Pluoronic F-127, cellulose, cyclodextrin, (2-Hydroxypropyl) - ⁇ -cyclodextrin, dextran (10, 40 and/or 70 kD) , polydextrose, maltodextrin, ficoll, gelatin, hydroxypropylmeth, sodium phosphate, potassium phosphate, ZnCl 2 , zinc, zinc oxide, sodium citrate, trisodium citrate
  • contemplated excipients which may be utilized in the aqueous pharmaceutical compositions of the patent application include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids such as phospholipids or fatty acids, steroids such as cholesterol, protein excipients such as serum albumin (human serum albumin) , recombinant human albumin, gelatin, casein, salt-forming counterions such sodium and the like.
  • a pharmaceutical container or vessel is used to hold the pharmaceutical formulation of any of conjugates of the patent application.
  • the vessel is a vial, bottle, pre-filled syringe, pre-filled orauto-injector syringe.
  • the liquid formula can be freeze-dried or drum-dryedto a form of cake or powder in a borosilicate vial or soda lime glass vial.
  • the solid powder can also be prepared by efficient spray drying, and then packed to a vial or a pharmaceutical container for storage and distribution.
  • the invention provides a method for preparing a formulation comprising the steps of: (a) lyophilizing the formulation comprising the conjugates, excipients, and a buffer system; and (b) reconstituting the lyophilized mixture of step (a) in a reconstitution medium such that the reconstituted formulation is stable.
  • the formulation of step (a) may further comprise a stabilizer and one or more excipients selected from a group comprising bulking agent, salt, surfactant and preservative as hereinabove described.
  • reconstitution media several diluted organic acids or water, i.e. sterile water, bacteriostatic water for injection (BWFI) or may be used.
  • the reconstitution medium may be selected from water, i.e.
  • sterile water bacteriostatic water for injection (BWFI) or the group consisting of acetic acid, propionic acid, succinic acid, sodium chloride, magnesium chloride, acidic solution of sodium chloride, acidic solution of magnesium chloride and acidic solution of arginine, in an amount from about 10 to about 250 mM.
  • BWFI bacteriostatic water for injection
  • a liquid pharmaceutical formulation of the conjugates of the patent application should exhibit a variety of pre-defined characteristics.
  • One of the major concerns in liquid drug products is stability, as the antibodies tend to form soluble and insoluble aggregates during manufacturing and storage.
  • various chemical reactions can occur in solution (deamidation, oxidation, clipping, isomerization etc. ) leading to an increase in degradation product levels and/or loss of bioactivity.
  • a conjugate in either liquid or loyphilizate formulation should exhibit a shelf life of more than 6 months at 25°C. More preferred a conjugate in either liquid or loyphilizate formulation should exhibit a shelf life of more than 12 months at 25°C.
  • liquid formulation should exhibit a shelf life of about 24 to 36 months at 2-8 °C and the loyphilizate formulation should exhibit a shelf life of about preferably up to 60 months at 2-8 °C. Both liquid and loyphilizate formulations should exhibit a shelf life for at least two years at -20 °C, or -70 °C.
  • the formulation is stable following freezing (e.g., -20°C, or -70 °C. ) and thawing of the formulation, for example following 1, 2 or 3 cycles of freezing and thawing.
  • Stability can be evaluated qualitatively and/or quantitatively in a variety of different ways, including evaluation of drug/antibody ratio and aggregate formation (for example using UV, size exclusion chromatography, by measuring turbidity, and/or by visual inspection) ; by assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometric analysis, or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS) , or HPLC-MS/MS; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS--C) analysis
  • Instability may involve any one or more of: aggregation, deamidation (e.g. Asn deamidation) , oxidation (e.g. Met oxidation) , isomerization (e.g. Asp isomeriation) , clipping/hydrolysis/fragmentation (e.g. hinge region fragmentation) , succinimide formation, unpaired cysteine (s) , N-terminal extension, C-terminal processing, glycosylation differences, etc.
  • deamidation e.g. Asn deamidation
  • oxidation e.g. Met oxidation
  • isomerization e.g. Asp isomeriation
  • clipping/hydrolysis/fragmentation e.g. hinge region fragmentation
  • a stable conjugate should also “retains its biological activity” in a pharmaceutical formulation, if the biological activity of the conjugate at a given time, e.g. 24 month, within about 20%, preferably about 10% (within the errors of the assay) of the biological activity exhibited at the time the pharmaceutical formulation was prepared as determined in an antigen binding assay, and/or in vitro, cytotoxic assay, for example.
  • the conjugate of the invention will be supplied as solutions or as a lyophilized solid that can be redissolved in sterile water for injection.
  • suitable protocols of conjugate administration are as follows. Conjugates are given dayly, weekly, biweekly, triweekly, once every four weeks or monthly for 8 ⁇ 108 weeks as an i.v. bolus. Bolus doses are given in 50 to 1000 ml of normal saline to which human serum albumin (e.g. 0.5 to 1 mL of a concentrated solution of human serum albumin, 100 mg/mL) can optionally be added. Dosages will be about 50 ⁇ g to 20 mg/kg of body weight per week, i.v.
  • Examples of medical conditions that can be treated according to the in vivo or ex vivo methods of killing selected cell populations include malignancy of any types of cancer, autoimmune diseases, graft rejections, and infections (viral, bacterial or parasite) .
  • the amount of a conjugate which is required to achieve the desired biological effect will vary depending upon a number of factors, including the chemical characteristics, the potency, and the bioavailability of the conjugates, the type of disease, the species to which the patient belongs, the diseased state of the patient, the route of administration, all factors which dictate the required dose amounts, delivery and regimen to be administered.
  • the conjugates of this invention may be provided in an aqueous physiological buffer solution containing 0.1 to 10%w/v conjugates for parenteral administration.
  • Typical dose ranges are from 1 ⁇ g/kg to 0.1 g/kg of body weight daily; weekly, biweekly, triweekly, or monthly, a preferred dose range is from 0.01 mg/kg to 25 mg/kg of body weight weekly, biweekly, triweekly, or monthly, an equivalent dose in a human.
  • the preferred dosage of drug to be administered is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, the formulation of the compound, the route of administration (intravenous, intramuscular, or other) , the pharmacokinetic properties of the conjugates by the chosen delivery route, and the speed (bolus or continuous infusion) and schedule of administrations (number of repetitions in a given period of time) .
  • a hyaluronidase (HAase) is preferably adminstered together with the conjugates.
  • the hyaluronidase here is used as an aid in helping patient body absorb the injected conjugates.
  • the hyaluronidase is synergistically used 20 -200 unit doses, preferably in 60 –160 unit doses.
  • the conjugates of the present invention are also capable of being administered in unit dose forms, wherein the term “unit dose” means a single dose which is capable of being administered to a patient, and which can be readily handled and packaged, remaining as a physically and chemically stable unit dose comprising either the active conjugate itself, or as a pharmaceutically acceptable composition, as described hereinafter.
  • unit doses for humans range from 1 mg to 3000 mg per day, or per week, per two weeks (biweekly) , triweekly, or per month.
  • the unit dose range is from 1 to 500 mg administered one to four times a month and even more preferably from 1 mg to 100 mg, once a week, or once a biweek, or once a triweek.
  • Conjugatess provided herein can be formulated into pharmaceutical compositions by admixture with one or more pharmaceutically acceptable excipients.
  • Such unit dose compositions may be prepared for use by oral administration, particularly in the form of tablets, simple capsules or soft gel capsules; or intranasally, particularly in the form of powders, nasal drops, or aerosols; or dermally, for example, topically in ointments, creams, lotions, gels or sprays, or via trans-dermal patches.
  • compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in Remington: The Science and Practice of Pharmacy, 21 th ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2005.
  • the formulations include pharmaceutical compositions in which a compound of the present invention is formulated for oral or parenteral administration.
  • tablets, pills, powders, capsules, troches and the like can contain one or more of any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, or gum tragacanth; a diluent such as starch or lactose; a disintegrant such as starch and cellulose derivatives; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, or methyl salicylate.
  • a binder such as microcrystalline cellulose, or gum tragacanth
  • a diluent such as starch or lactose
  • a disintegrant such as starch and cellulose derivatives
  • a lubricant such as magnesium stearate
  • a glidant such
  • Capsules can be in the form of a hard capsule or soft capsule, which are generally made from gelatin blends optionally blended with plasticizers, as well as a starch capsule.
  • dosage unit forms can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents.
  • Other oral dosage forms syrup or elixir may contain sweetening agents, preservatives, dyes, colorings, and flavorings.
  • the active compounds may be incorporated into fast dissolve, modified-release or sustained-release preparations and formulations, and wherein such sustained-release formulations are preferably bi-modal.
  • Preferred tablets contain lactose, cornstarch, magnesium silicate, croscarmellose sodium, povidone, magnesium stearate, or talc in any combination.
  • Liquid preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • the liquid compositions may also include binders, buffers, preservatives, chelating agents, sweetening, flavoring and coloring agents, and the like.
  • Non-aqueous solvents include alcohols, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate.
  • Aqueous carriers include mixtures of alcohols and water, buffered media, and saline.
  • biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of the active compounds.
  • Intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer’s dextrose, and the like.
  • Other potentially useful parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • formulations for inhalation which include such means as dry powder, aerosol, or drops. They may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
  • Formulations for buccal administration include, for example, lozenges or pastilles and may also include a flavored base, such as sucrose or acacia, and other excipients such as glycocholate.
  • Formulations suitable for rectal administration are preferably presented as unit-dose suppositories, with a solid based carrier, such as cocoa butter, and may include a salicylate.
  • Formulations for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.
  • Carriers which can be used include petroleum jelly, lanolin, polyethylene glycols, alcohols, or their combinations.
  • Formulations suitable for transdermal administration can be presented as discrete patches and can be lipophilic emulsions or buffered, aqueous solutions, dissolved and/or dispersed in a polymer or an adhesive.
  • a pharmaceutical composition comprising a therapeuticcally effective amount of the conjugate of Formula (I) , (II) , (III) , or any conjugates described through the present patent can be coadministered with the other therapeutic agents such as the chemotherapeutic agent, the radiation therapy, immunotherapy agents, autoimmune disorder agents, anti-infectious agents or the other conjugates for synergistically effective treatment or prevention of a cancer, or an autoimmune disease, or an infectious disease.
  • the other therapeutic agents such as the chemotherapeutic agent, the radiation therapy, immunotherapy agents, autoimmune disorder agents, anti-infectious agents or the other conjugates for synergistically effective treatment or prevention of a cancer, or an autoimmune disease, or an infectious disease.
  • administered refers to administering one or more additional therapeutic agents and the antibody or ADC described herein, or the antibody or ADC-containing composition, sufficiently close in time such that the antibody or ADC can enhance the effect of one or more additional therapeutic agents, or vice versa.
  • the antibody or ADC or the composition containing the same may be administered first, and the one or more additional therapeutic agents may be administered second, or vice versa.
  • the antibody or ADC or composition containing the same may be administered in combination with other agents (e.g., as an adjuvant) for the treatment or prevention of multiple myeloma.
  • the antibody or ADC or antibody or ADC-containing composition can be used in combination with at least one other anticancer agent including, for example, any suitable chemotherapeutic agent known in the art, ionization radiation, small molecule anticancer agents, cancer vaccines, biological therapies (e.g., other monoclonal antibodies, cancer-killing viruses, gene therapy, and adoptive T-cell transfer) , and/or surgery.
  • the synergisticdrugs or radiation therapy can be administered prior or subsequent to administration of a conjugate, in one aspect at least an hour, 12 hours, a day, a week, biweeks, triweeks, a month, in further aspects several months, prior or subsequent to administration of a conjugate of the invention.
  • the synergistic agents are preferably selected from one or several of the following drugs: Abatacept, Abiraterone acetate, Abraxane, Acetaminophen/hydrocodone, Acalabrutinib, aducanumab, Adalimumab, ADXS31-142, ADXS-HER2, Afatinibdimaleate, Aldesleukin, Alectinib, Alemtuzumab, Alitretinoin, ado-trastuzumab emtansine, Amphetamine/dextroamphetamine, Anastrozole, Aripiprazole, anthracyclines, Aripiprazole, Atazanavir, Atezolizumab, Atorvastatin, Avelumab, Axicabtageneciloleucel, Axitinib, Belinostat, BCG Live, Bevacizumab, Bexarotene, Blinatumo
  • the disclosure also provides a composition
  • a composition comprising the above-described antibody or antibody-drug conjugateand a pharmaceutically acceptable (e.g., physiologically acceptable) carrier.
  • a pharmaceutically acceptable carrier e.g., physiologically acceptable
  • Any suitable carrier known in the art can be used within the context of the invention. The choice of carrier will be determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition.
  • the composition optionally may be sterile.
  • the compositions can be generated in accordance with conventional techniques described in, e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2001) .
  • the composition of this invention desirably comprises the antibody or ADCsin an amount that is effective to treat or prevent multiple myeloma.
  • the disclosure provides a method of killing multiple myeloma cells, which comprises contacting multiple myeloma cells that express BCMAwith the antibody or ADCs described herein, or a composition comprising the antibody or ADC described herein, whereby the antibody orADCsbinds to BCMAon the multiple myeloma cells and kills the multiple myeloma cells.
  • the disclosure also provides use of the antibody or ADC described herein, or the composition comprising the antibody or ADC, in the manufacture of a medicament for treating multiple myeloma.
  • multiple myeloma also known as plasma cell myeloma or Kahler's disease
  • plasma cell myeloma is a cancer of plasma cells, which are a type of white blood cell normally responsible for the production of antibodies (Raab et al., Lancet, 374: 324-329 (2009) ) .
  • Multiple myeloma affects 1 ⁇ 4 per 100,000 people per year. The disease is more common in men, and for yet unknown reasons is twice as common in African Americans as it is in Caucasian Americans. Multiple myeloma is the least common hematological malignancy (14%) and constitutes 1%of all cancers.
  • Treatment of multiple myeloma typically involves high-dose chemotherapy followed by hematopoietic stem cell transplantation (allogenic or autologous) ; however, a high rate of relapse is common in multiple myeloma patients that have undergone such treatment.
  • BCMA is highly expressed by multiple myeloma cells.
  • BCMA also is expressed on multiple myeloma stem cells.
  • the disclosure provides a method of killing multiple myeloma stem cells, which comprises contacting multiple myeloma stem cells that express BCMA with the antibody-drug conjugatedescribed herein, or a composition comprising the ADC described herein, whereby the antibody-drug conjugatebinds to BCMAon the multiple myeloma stem cells and kills the multiple myeloma stem cells.
  • myeloma stem cells can be identified in the bone marrow of multiple myeloma patients by their surface expression of CD19 and lack of CD138 surface expression (see, e.g., Matsui et al., Blood, 103: 2332-6 (2004) ) . These cells are uniquely clonogenic and engraft immunodeficient mice, whereas the myeloma plasma cells, defined as CD138+CD19-, do not. Multiple myeloma stem cells also are resistant to current therapies (Matsui et al., Cancer Res., 68: 190-7 (2008) ) .
  • the invention provides a method of treating a patient having or at risk of having a cancer that expresses BCMA comprising administering to the patient an effective regime of the BCMA antibody or the BCMA ADC as described above.
  • the cancer is a hematological cancer.
  • the hematological cancer is a myeloma, leukemia or a lymphoma.
  • the hematological cancer is multiple myeloma.
  • the hematological cancer is non-Hodgkin's lymphoma (NHL) or Hodgkin's lymphoma.
  • the hematological cancer is myelodysplastic syndromes (MDS) , myeloproliferative syndromes (MPS) , Waldenstrom's macroglobulinemia or Burkett's lymphoma.
  • the terms “treatment, “ “treating, “ and the like refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect is therapeutic, i.e., the effect partially or completely cures a disease and/or adverse symptom attributable to the disease.
  • the inventive method comprises administering a "therapeutically effective amount" of the antibody or ADC or the composition comprising the antibody or ADC and a pharmaceutically acceptable carrier.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • the therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or ADC to elicit a desired response in the individual.
  • a therapeutically effective amount of the ADC of the invention is an amount which binds to BCMAon multiple myeloma cells and destroys them.
  • Apharmacologic and/or physiologic effect of treatment may be prophylactic, i.e., the effect completely or partially prevents a disease or symptom thereof.
  • the inventive method comprises administering a "prophylactically effective amount" of the ADC or a composition comprising the ADC to a mammal that is predisposed to multiple myeloma.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of disease onset) .
  • Therapeutic or prophylactic efficacy can be monitored by periodic assessment of treated patients.
  • the ADC described herein inhibits or suppresses proliferation of BCMA-expressing myeloma cells by at least about 10% (e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%) .
  • Cell proliferation can be measured using any suitable method known in the art, such as measuring incorporation of labeled nucleosides (e.g., 3H-thymidine or bromodeoxyuridine Brd (U) ) into genomic DNA (see, e.g., Madhavan, H.N., J. Stem Cells Regen. Med., 3 (1) : 12-14 (2007) ) .
  • labeled nucleosides e.g., 3H-thymidine or bromodeoxyuridine Brd (U)
  • the invention of the BCMA antibody and BCMA ADCsfurther provides a method of treating a patient having or at risk of having an immune disorder mediated by immune cells expressing BCMA comprising administering to the patient an effective regime of any of the above described antibodies or ADCs.
  • the disorder is a B cell mediated disorder.
  • the immune disorder is rheumatoid arthritis, systemic lupus E (SLE) , Type I diabetes, asthma, atopic dermitus, allergic rhinitis, thrombocytopenic purpura, multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, and graft versus host disease.
  • SLE systemic lupus E
  • the invention of the BCMA antibody and the BCMA ADCs further provides a method of treating a patient having or at risk of having a cancer, an autoimmune disease, an infectious disease, viral disease or a pathogenic infection, through administering to the patient an effective regime of any of the above described antibodies or ADCs, or any of the above described antibodies or ADCs concurrently withthe other therapeutic agents such as the chemotherapeutic agent, the radiation therapy, immunotherapy agents, autoimmune disorder agents, anti-infectious agents or the other conjugates.
  • the targeted cancer includes, but are not limited, Adrenocortical Carcinoma, Anal Cancer, Bladder Cancer, Brain Tumor (Adult, Brain Stem Glioma, Childhood, Cerebellar Astrocytoma, Cerebral Astrocytoma, Ependymoma, Medulloblastoma, Supratentorial Primitive Neuroectodermal and Pineal Tumors, Visual Pathway and Hypothalamic Glioma) , Breast Cancer, Carcinoid Tumor, Gastrointestinal, Carcinoma of Unknown Primary, Cervical Cancer, Colon Cancer, Endometrial Cancer, Esophageal Cancer, Extrahepatic Bile Duct Cancer, Ewings Family of Tumors (PNET) , Extracranial Germ Cell Tumor, Eye Cancer, Intraocular Melanoma, Gallbladder Cancer, Gastric Cancer (Stomach) , Germ Cell Tumor, Extragonadal, Gestational Trophoblastic Tumor, Head and Neck Cancer
  • the autoimmune disease includes, but are not limited, Achlorhydra Autoimmune Active Chronic Hepatitis, Acute Disseminated Encephalomyelitis, Acute hemorrhagic leukoencephalitis, Addison’s Disease, Agammaglobulinemia, Alopecia areata, Amyotrophic Lateral Sclerosis, Ankylosing Spondylitis, Anti-GBM/TBM Nephritis, Antiphospholipid syndrome, Antisynthetase syndrome, Arthritis, Atopic allergy, Atopic Dermatitis, Autoimmune Aplastic Anemia, Autoimmune cardiomyopathy, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome, Autoimmune peripheral neuropathy, Autoimmune pancreatitis, Autoimmune polyendocrine syndrome Types I, II, & III, Autoimmune progesterone dermatitis, Autoimmune thro
  • the infectious disease includes, but are not limited to, Acinetobacter infections, Actinomycosis, African sleeping sickness (African trypanosomiasis) , AIDS (Acquired immune deficiency syndrome) , Amebiasis, Anaplasmosis, Anthrax, Arcano-bacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereus infection, Bacterial pneumonia, Bacterial vaginosis, Bacteroides infection, Balantidiasis, Baylisascaris infection, BK virus infection, Black piedra, Blastocystis hominis infection, Blastomycosis, Cambodian hemorrhagic fever, Borrelia infection, Botulism (and Infant botulism) , Brazilian hemorrhagic fever, Brucellosis, Burkholderia infection, Buruli ulcer, Calicivirus infection (N
  • the pathogenic strain includes, but are not limit, Acinetobacter baumannii, Actinomyces israelii, Actinomyces gerencseriae and Propionibacterium propionicus, Trypanosoma brucei, HIV (Human immunodeficiency virus) , Entamoeba histolytica, Anaplasma genus, Bacillus anthracis, Arcanobacteriumhaemolyticum, Junin virus, Ascaris lumbricoides, Aspergillus genus, Astroviridae family, Babesia genus, Bacillus cereus, multiple bacteria, Bacteroides genus, Balantidium coli, Baylisascaris genus, BK virus, Piedraiahortae, Blastocystis hominis, Blastomyces dermatitides, Machupo virus, Borrelia genus, Clostridium botulinum, Sabia, Brucella genus, usually Burkholderi
  • the pathogenic viruse includes, but not by limitation: Poxyiridae, Herpesviridae, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, Oncovirus [such as, HBV (Hepatocellular carcinoma) , HPV (Cervical cancer, Anal cancer) , Kaposi’s sarcoma-associated herpesvirus (Kaposi’s sarcoma) , Epstein-Barr virus (Nasopharyngeal carcinoma, Burkitt’s lymphoma, Primary central nervous system lymphoma
  • the present invention also concerns pharmaceutical compositions comprising the BCMA antibodyor ADCs of the invention together with a pharmaceutically acceptable carrier, diluent, or excipient for treatment of cancers, infections or autoimmune disorders.
  • a pharmaceutically acceptable carrier diluent, or excipient for treatment of cancers, infections or autoimmune disorders.
  • the method for treatment of cancers, infections and autoimmune disorders can be practiced in vitro, in vivo, or ex vivo.
  • in vitro uses include treatments of cell cultures in order to kill all cells except for desired variants that do not express the target antigen; or to kill variants that express undesired antigen.
  • ex vivo uses include treatments of hematopoietic stem cells (HSC) prior to the performance of the transplantation (HSCT) into the same patient in order to kill diseased or malignant cells.
  • HSC hematopoietic stem cells
  • the bone marrow cells are washed with medium containing serum and returned to the patient by i.v. infusion according to known methods.
  • the treated marrow cells are stored frozen in liquid nitrogen using standard medical equipment.
  • NMR spectra were recorded on Zhongke-niujin WNMR-I 400 MHz instrument at the Department of Chemistry of Zhejiang Sci-Tech University. Chemical shifts ( ⁇ ) are reported in parts per million (ppm) referenced to tetramethylsilane at 0.00 and coupling constants (J) are reported in Hz.
  • the elemental analysis of C, H, and/or N was provided by the Department of Chemistry of Zhejiang Sci-Tech University and conducted on Elementar UNICUBE. Quantitative analysis of metal atoms was performed on Agilent ICPOES 730 ICP-MS.
  • Example 1 The generation of a monoclonal antibody directed against B-cell maturation antigen (BCMA) .
  • BCMA B-cell maturation antigen
  • mice were immunized over a course of 13 days at intervals of 2-3 days. For each round of immunization, mice were first anesthetized with isoflurane. The immunogen was emulsified in complete or incomplete Freund's adjuvant. Gold adjuvant (Sigma-Aldrich) and injected bilaterally at multiple sites. Test bleeds were collected on day 13 and assayed in antigen ELISA.
  • mice with good serum titers were given a pre-fusion boost intraperitoneally and sacrificed on day 17.
  • Spleen cells were harvested and fused to myeloma cell line P3-X63-Ag8.653 following the polyethylene glycol fusion method (Roche Diagnostics) to generate stable hybridomas.
  • Anti-BCMA-specific hybridomas were identified by screening the hybridoma supernatants in direct binding ELISA followed by FACS on BCMA-expressing RPMI-8226-BCMA cells. Positive hybridomas were further tested for their ability to bind, internalize RPMI-8226-BCMA cells in vitro and by FACS binding to BCMA expressed on cell lines.
  • cloneBCMA-A2-6H4-5D2 After selection and subclone, cloneBCMA-A2-6H4-5D2were selected, antibody binding affinity were determined by Elisa assay along with anti-BCMA antibody J6M0 (described in US. Pat. No. 9,273,141, called belantamab or DXA009B in the application) , results show in Fig 1.
  • a deposit at China Center for Type Culture Collection (CCTCC) was made on June23, 2022 under the Budapest Treaty.
  • the CCTCC is located at Wuhan University, Wuhan City, Hubei, Post code 430000, P.R. China.
  • the CCTCC deposit was assigned accession number of CCTCC C2022188.
  • amino acids sequences of the heavy and light chain variable regions of the monoclonal antibody BCMA-A2-6H4-5D2 are shown in Table 1.
  • Example 2 The generation of humanized monoclonal antibodies of BCMA-A2-6H4-5D2.
  • Chimeric antibody c5D2 HC (SEQ ID NO: 13) constructed by fusion VH of BCMA-A2-6H4-5D2 (SEQ ID NO: 10) with human IgG1 HC constant domain (SEQ ID NO: 12, which is encoded by the SEQ ID NO: 24)
  • Chimeric antibody c5D2 LC (SEQ ID NO: 15) constructed by fusionVL of BCMA-A2-6H4-5D2 (SEQ ID NO: 11) with human Kappa LC constant domain (SEQ ID NO: 14, which is encoded by the SEQ ID NO: 26) .
  • Humanized antibody hu5D2 HC (SEQ ID NO: 8) generated by substitution of corresponding Amino Acid of 5D2 with human germine line gene Amino Acid
  • Humanized antibody hu5D2 LC (SEQ ID NO: 9) generated by substitution of corresponding Amino Acid of 5D2 with human germine line gene Amino Acid.
  • the affinity of hu5D2 showed in Fig 2.
  • amino acids sequences of the heavy and light chain variable regions of the monoclonal antibody c5D2 and hu5D2 are shown in Table 2.
  • Example 3 Atridationalmethod of producing an antibody-drug conjugate (ADC) comprising a BCMA monoclonal antibody conjugated to a cytotoxin having a terminal of maleimido group.
  • ADC antibody-drug conjugate
  • the hu5D2 monoclonal antibody was conjugated to a cytotoxin having a terminal of maleimido group. Specifically, purified antibody was incubated with a 3.2 –4.2 molar excess of the reducing agent TCEP (Tris (2-carboxyethyl) phosphine) in PBS pH 7.2, 1 mM EDTA (Ethylenediamine tetraaceticacid) for 1 hours at 37°C.
  • TCEP Tris (2-carboxyethyl) phosphine
  • the conjugation process may result in 0.1 to 10%of aggregate formation.
  • Macromolecular aggregates, conjugation reagents, including cysteine quenched paylaods, can be removed using ceramic hydroxyapatite Type II chromatography (CHT) as described e.g. Thompson et al., J. Control Release, 236: 100-116 (2016) .
  • CHT ceramic hydroxyapatite Type II chromatography
  • the ADCs were optionally formulated in 25 mM Histidine-HCl, 7%sucrose, 0.02%polysorbate-20 or 80, pH 6.
  • Hydrophobic interaction chromatography was used to assess conjugation and drug load distribution, and was performed using a butyl-non porous resin (NPR) column (4.6 mm ID x3.5 cm, 2.5 ⁇ m, Tosoh Bioscience) .
  • the mobile phase A was composed of 25 mM Tris-HCl, 1.5 M (NH 4 ) 2 SO 4 , pH 8.0; and the mobile phase B was composed of 25 mM Tris-HCl and 5%isopropanol, pH 8.0.100 ⁇ L of antibodies or ADCs at a concentration of 1 mg/mL were loaded and eluted at a flow rate of 1 mL/min with a gradient of 5%B to 100%B over 10 -30 min.
  • rRP-HPLC Reduced reverse phase chromatography
  • the antibodies and ADCs were reduced at 37°C. for 20 minutes using 42 mM dithiothreitol (DTT) in PBS (pH 7.2) .
  • 10 ⁇ g of reduced antibodies or ADCs were loaded onto a polymeric reverse phase media (PLRP-S) 1000 A column (2.1 x 50 mm) (Agilent Technologies, Santa Clara, Calif. ) and eluted at 80°C.
  • PLRP-S polymeric reverse phase media
  • Conjugation at the heavy and light chains and drug/antibody ratios were determined by reduced liquid chromatography mass spectrometry analysis (rLCMS) performed on an Agilent 1290 series uHPLC coupled to an Agilent 6230 TOF (Agilent Technologies, Santa Clara, Calif. ) . 2 ⁇ g of reduced antibodies or ADCs were loaded onto a ZORBAX.
  • Example 4 A method of production of BCMA expression cell lines.
  • Stable cell lines were developed by transfecting RPMI-8226 cells with either a full-length BCMA clone or an empty vector coexpress GFP protein. Flow cytometry confirmed positive expression of BCMA on the surface of the BCMA transfected (RPMI-8226-BCMA) . These cell lines were subsequently used as a tool to confirm the specificity of cloned BCMA antibodies.
  • Example 5 The binding affinity of monoclonal BCMA antibodies and ADCs described herein to soluble and membrane-bound BCMA..
  • Elisa assay Binding of BCMA-A2-6H4-5D2, hu5D2 and c5D2 to soluble BCMA was determined by using Elisa assay. Elisa assay were performed coating 1 ⁇ g/ml soluble BCMA, 50 ⁇ L /Well for 1 hour at 37 °C, followed by blocking with PBS+2%BSA. A range of antibody concentrations were diluted, and added to pre-coated Elisa plate for incubation for 1 hour at 37 °C, followed by 3 times PBST wash (BioTek 405) and then 50 ⁇ L /Well goat Anti-Human IgG (Fab specific) -Peroxidase (Sigma-Aldrich) (1: 20000 diluted ) were added for detection. Before detection, plates were washed by PBST for 3 times, then TMB were added and stopped by addition of 2M H 2 SO 4 . The results of this experiment are shown in Fig. 2.
  • Binding of hu5D2, c5D2 and hu5D2-tub196 to membrane-bound human BCMA was evaluated using flow cytometry in multiple myeloma cell lines that endogenously express BCMA (NCI-H929) . Binding assays were performed by incubating the anti-BCMA antibodies with 200,000 cells for 30 minutes at 4 °C, followed by two washes with PBS+2%FBS (FACS Buffer) . A range of antibody concentrations were evaluated using an 11-point, 4-fold dilution series.
  • Example 6 The methods of killing multiple myeloma cells in vitro using the antibody-drug conjugates.
  • the antibody-drug conjugates were diluted to a 10x stock (100 ⁇ g/mL) in RPMI+10%FBS. Treatments were then serially diluted 1: 10 in RPMI+10%FBS. 20 ⁇ L of this series was added to the cells in triplicate, resulting in a 8-point dose curve of antibody-drug conjugate ranging from 10 ⁇ g/mL at the highest concentration to 0 vg/mL at the lowest. Plates were incubated at 37°C., 5%CO 2 for 96 hours. At the end of the incubation period, 10 ⁇ L of the Substrate Solution was added to each well.
  • the absorbance at 450 nm was measured using a SpectraMax i3x plate reader (Molecular Device, USA) . Data were analyzed and graphed using GraphPad Prismor Excel software, and the half-maximal inhibitory concentration (IC 50 ) was determined. The results of this experiment are shown in Fig. 4A, 4B, 4C, 4D.
  • SampleInformation Recombinant humanized anti-BCMA monoclonal antibody (DXA009 DS) , Batch number is 009A2201B (produced by the applicant of this patent application: Hangzhou DAC Biotechnology Co., Ltd) .
  • Sample preparation Glycospeptide EEQYNSTYR (SEQ ID NO: 34) with glycans attached at asparagine position.
  • Recombinant humanized anti-BCMA monoclonal antibody (DXA009 DS, Batch number is 009A2201B) , was denatured and reduced with 6M Urea, 10mM dithiothreitol at 56°C for about 40 min) , alkylated (about 30mM Iodoacetamide, 40 min in the dark at room temperature) , diluted in 50mM NH 4 HCO 3 and digested with Trypsin (1/50, enzyme/substrate weight ratio, 4h, 37 °C) .
  • N-glycoside is confirmed at Asn (N) -300.
  • G Galactose
  • F Fucose
  • Man5 Manose5
  • GlcNAc N-Acetylglucosamine
  • LC system Waters ACQUITY UPLC H-Class System, Detector: ACQUITY UPLC TUV, Absorption Wavelength: 214nm; Trap Column: ACQUITY UPLC BEH C18 1.7 ⁇ m 2.1 ⁇ 100 mm Column; Mobile phase A: 0.1%formic acid (FA) in water, Mobile phase B: 0.1%formic acid (FA) in ACN. Performed the chromatographic separation at a flow rate of 0.25 ml/min using a linear gradient of mobile phase B (ACN with 0.1%FA) from 1%to 40%over 95 min., followed by from 40%to 80%for 10 min and then 80%to 80%for 5 min.
  • ACN 0.1%formic acid
  • MS conditions MS system: Waters Xevo-G2XS Q-TOF; Ionization mode: ESI positive, Sensitivity Mode; Data Acquisition: MS E ; Mass Range: m/z 100-2500 Da; Informatics: Perform the data analysis using UNIFI V1.8.2.169 Software (Waters) .
  • Example 8 Reduced Molecular Weight and DAR Analysis for the DeglycosylatedBCMA-Tub ADCs by LC-MS.
  • Sample preparation Reductionof an ADC (e.g. (DXC009 DP) with 5mM dithiothreitol at 37°Cfor about 2 h, followed by a deglycosylation stepwith PNGase F at 37°C overnight generated six fragments as illustrated in Fig. 6 and 7.
  • HC and LC existed as naked or conjugated forms carrying up to 3 payloads.
  • the masses of each ADC fragments and the average DARs of the ADC as illustrated in Table 5. The following equation was used for average DAR calculation for conventional conjugated ADC.
  • AverageDAR L1/ (L0+L1) x 2+H1/ (H0+H1+H2+H3) x 2+H2/ (H0+H1+H2+H3) x 2+H3/ (H0+H1+H2+H3) x 2.
  • UPLC system Waters ACQUITY UPLC H-Class System; Detector: ACQUITY UPLC TUV; Absorption Wavelength: 280nm; Trap Column: ACQUITY UPLC C4 1.7 ⁇ m 2.1 x 50mm Column; Mobile phase A: 0.1%formic acid (FA) in water, Mobile phase B: 0.1%formic acid (FA) in ACN; Performed the chromatographic separation at a flow rate of 0.4 ml/min using a linear gradient of mobile phase B (ACN with 0.1%FA) from 5%to 25%for2 min, followed by 25%to 45%for 8 min, then 45%to 85%for 2 min.
  • ACN 0.1%formic acid
  • MS conditions MS system: Waters Xevo-G2XS Q-TOF; Ionization mode: ESI positive; Mass Range: m/z 500-4000 Da. Informatics: the data analysis using UNIFI V1.8.2.169 Software (Waters) .
  • Example 9 Drug Conjugation Site Analysis for the BCMA-ADCs by LC-MS.
  • Samplepreparation Recombinant humanized anti-BCMA monoclonal antibody-Tubulysin B conjugate (e.g. DXC009 DP) , Batch number is 22030251, Pack size is 100 mg/bottle, manufactured by Hangzhou DAC Biotechnology Co., Ltd.
  • ADC samples were denatured and reduced (6M Urea, 10mM dithiothreitol at 56°C for about 40 min) , alkylated (about 30mM Iodoacetamide, 40 min in the dark at room temperature) , diluted in 50mM HEPES and digested with trypsin (1/50, enzyme/substrate weight ratio, 4h, 37 °C) .
  • the drug-loaded peptides of ADC as illustrated in Table 6.
  • LC system Waters ACQUITY UPLC H-Class System; Detector: ACQUITY UPLC TUV, Absorption Wavelength: 214nm; Trap Column: ACQUITY UPLC C18 1.7 ⁇ m 2.1 ⁇ 100 mm Column; Mobile phase A: 0.1%formic acid (FA) in water, Mobile phase B:0.1%formic acid (FA) in ACN; Perform the chromatographic separation at a flow rate of 0.2 ul/min using a linear gradient of mobile phase B (ACN with 0.1%FA) from 1%to 40%over 95 min., followed by 40%to 80%for 15 min.;
  • MS conditions MS system: Waters Xevo-G2XS Q-TOF; Ionization mode: ESI positive, Sensitivity Mode; Data Acquisition: MS E ; Mass Range: m/z 100-2500 Da; Informatics: Perform the data analysis using UNIFI V1.8.2.169 Software (Waters) .
  • Example 16 Synthesis of di-tert-butyl 4, 4'- ( (2, 3-bis ( (4R, 7S) -1, 3-dioxo-1, 3, 3a, 4, 7, 7a-hexahydro-2H-4, 7-epoxyisoindol-2-yl) succinyl) bis (azanediyl) ) dibutyrate (10) .
  • Example 17 Synthesis of di-tert-butyl 4, 4'- ( (2, 3-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) succinyl) bis (azanediyl) ) dibutyrate (11) .
  • racemic mixture (61 g) as a white solid was collected, and a racemic/meso mixture was also recovered from the mother liquid, which could be re-processed to afford a clean meso compound (2 g) and other racemic/meso mixture (15 g) .
  • Example 20 Synthesis of di-tert-butyl 4, 4'- ( ( (2R, 3R) -2, 3-bis ( ( (benzyloxy) carbonyl) amino) succinyl) bis (azanediyl) ) dibutyrate (14) .
  • tert-butyl aminobutyrate hydrochloride 31 g, 0.151 mol
  • HATU 86.12 g, 0.226 mol
  • triethylamine 42 mL, 0.302 mmol
  • Example 21 Synthesis of di-tert-butyl 4, 4'- ( ( (2R, 3R) -2, 3-diaminosuccinyl) bis (azanediyl) ) dibutyrate (15) .
  • Example 23 Synthesis of di-tert-butyl 4, 4'- ( ( (2R, 3R) -2, 3-bis ( (4R, 7S) -1, 3-dioxo-1, 3, 3a, 4, 7, 7a-hexahydro-2H-4, 7-epoxyisoindol-2-yl) succinyl) bis (azanediyl) ) dibutyrate (17) .
  • Example 24 Synthesis of di-tert-butyl 4, 4'- ( ( (2R, 3R) -2, 3-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) succinyl) bis (azanediyl) ) dibutyrate (18) .
  • Example 25 Synthesis of 4, 4'- ( ( (2R, 3R) -2, 3-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) succinyl) bis (azanediyl) ) dibutyric acid (19) .
  • Example 26 Synthesis of tert-butyl (S) - (37- ( ( (benzyloxy) carbonyl) amino) -31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azaoctatriacontan-38-oyl) glycinate (21) .
  • Example 27 Synthesis of tert-butyl (S) - (37-amino-31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azaoctatriacontan-38-oyl) glycinate (22) .
  • Example 28 Synthesis of di-tert-butyl (5S, 13S, 14S, 22S) -13, 14-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -4, 7, 12, 15, 20, 23-hexaoxo-5, 22-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 6, 11, 16, 21, 24-hexaazahexacosanedioate (23) .
  • Example 29 Synthesis of (5S, 13S, 14S, 22S) -13, 14-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -4, 7, 12, 15, 20, 23-hexaoxo-5, 22-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 6, 11, 16, 21, 24-hexaazahexacosanedioic acid (24) .
  • Example 31 Synthesis of tert-butyl (S) - (S) - (37- ( ( (benzyloxy) carbonyl) amino) -31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azaoctatriacontan-38-oyl) glycylglycinate (26) .
  • Example 32 Synthesis of tert-butyl (S) - (S) - (37-amino-31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azaoctatriacontan-38-oyl) glycylglycinate (27) .
  • Example 33 Synthesis of di-tert-butyl (8S, 16S, 17S, 25S) -16, 17-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -4, 7, 10, 15, 18, 23, 26, 29-octaoxo-8, 25-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 6, 9, 14, 19, 24, 27, 30-octaazadotriacontanedioate (28) .
  • Example 34 Synthesis of (8S, 16S, 17S, 25S) -16, 17-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -4, 7, 10, 15, 18, 23, 26, 29-octaoxo-8, 25-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 6, 9, 14, 19, 24, 27, 30-octaazadotriacontanedioic acid (29) .
  • Example 36 Synthesis of tert-butyl (S) - (37- ( ( (benzyloxy) carbonyl) amino) -31, 38-dioxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32, 39-diazatritetracontan-43-oyl) glycinate (32) .
  • Example 37 Synthesis of tert-butyl (S) - (37-amino-31, 38-dioxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32, 39-diazatritetracontan-43-oyl) glycinate (33) .
  • Example 38 Synthesis of di-tert-butyl (10S, 18S, 19S, 27S) -18, 19-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -4, 9, 12, 17, 20, 25, 28, 33-octaoxo-10, 27-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 8, 11, 16, 21, 26, 29, 34-octaazahexatriacontanedioate (34) .
  • Example 39 Synthesis of (10S, 18S, 19S, 27S) -18, 19-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -4, 9, 12, 17, 20, 25, 28, 33-octaoxo-10, 27-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 8, 11, 16, 21, 26, 29, 34-octaazahexatriacontanedioic acid (35) .
  • Example 41 Synthesis of tert-butyl ( (benzyloxy) carbonyl) glycylglycylglycinate (38) .
  • Example 42 Synthesis of tert-butyl glycylglycylglycinate (39) .
  • Example 43 Synthesis of tert-butyl (S) - (S) - (S) - (S) - (37- ( ( (benzyloxy) carbonyl) amino) -31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azaoctatriacontan-38-oyl) glycylglycylglycinate (40) .
  • Example 44 Synthesis of tert-butyl (S) - (S) - (S) - (S) - (37-amino-31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azaoctatriacontan-38-oyl) glycylglycylglycinate (41) .
  • Example 45 Synthesis of di-tert-butyl (11S, 19S, 20S, 28S) -19, 20-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -4, 7, 10, 13, 18, 21, 26, 29, 32, 35-decaoxo-11, 28-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 6, 9, 12, 17, 22, 27, 30, 33, 36-decaazaoctatriacontanedioate (42) .
  • Example 46 Synthesis of (11S, 19S, 20S, 28S) -19, 20-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -4, 7, 10, 13, 18, 21, 26, 29, 32, 35-decaoxo-11, 28-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 6, 9, 12, 17, 22, 27, 30, 33, 36-decaazaoctatriacontanedioic acid (43) .
  • Example 47 Synthesis of bis (perfluorophenyl) (11S, 19S, 20S, 28S) -19, 20-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -4, 7, 10, 13, 18, 21, 26, 29, 32, 35-decaoxo-11, 28-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 6, 9, 12, 17, 22, 27, 30, 33, 36-decaazaoctatriacontanedioate (44) .
  • Example 49 Synthesis of (S) - (S) - (37- ( ( (benzyloxy) carbonyl) amino) -31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azaoctatriacontan-38-oyl) glycylglycine (46) .
  • Example 50 Synthesis of tert-butyl ( (S) -37- ( ( (benzyloxy) carbonyl) amino) -31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azaoctatriacontan-38-oyl) glycylglycyl-L-alaninate (47) .
  • Example 51 Synthesis of tert-butyl ( (S) -37-amino-31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azaoctatriacontan-38-oyl) glycylglycyl-L-alaninate (48) .
  • Example 52 Synthesis of di-tert-butyl (2S, 11S, 19S, 20S, 28S, 37S) -19, 20-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -2, 37-dimethyl-4, 7, 10, 13, 18, 21, 26, 29, 32, 35-decaoxo-11, 28-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 6, 9, 12, 17, 22, 27, 30, 33, 36-decaazaoctatriacontanedioate (49) .
  • Example 53 Synthesis of (2S, 11S, 19S, 20S, 28S, 37S) -19, 20-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -2, 37-dimethyl-4, 7, 10, 13, 18, 21, 26, 29, 32, 35-decaoxo-11, 28-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 6, 9, 12, 17, 22, 27, 30, 33, 36-decaazaoctatriacontanedioic acid (50) .
  • Example 54 Synthesis of (2S, 3S) -2, 3-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N1, N4-bis ( (S) -37- ( (2- ( (2- ( ( (S) -1- ( ( (1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-1, 2, 3, 9, 10, 12, 13, 15-octahydrobenzo [de] pyrano [3', 4': 6, 7] indolizino [1, 2-b] quinolin-1-yl) amino) -1-oxopropan-2-yl) amino) -2-oxoethyl) amino) -2-oxoethyl) carbamoyl) -31, 39-dioxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32, 38-diazadotetracontan-42-yl
  • Example 55 Synthesis of tert-butyl ( (benzyloxy) carbonyl) glycylglycylglycylglycinate (52) .
  • Example 56 Synthesis of tert-butyl glycylglycylglycylglycinate (53) .
  • Example 57 Synthesis of tert-butyl (S) - (S) - (S) - (S) - (S) - (37- ( ( (benzyloxy) carbonyl) amino) -31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azaoctatriacontan-38-oyl) glycylglycylglycylglycinate (54) .
  • Example 58 Synthesis of tert-butyl (S) - (S) - (S) - (S) - (S) - (37-amino-31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azaoctatriacontan-38-oyl) glycylglycylglycylglycinate (55) .
  • Example 59 Synthesis of di-tert-butyl (14S, 22S, 23S, 31S) -22, 23-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -4, 7, 10, 13, 16, 21, 24, 29, 32, 35, 38, 41-dodecaoxo-14, 31-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 6, 9, 12, 15, 20, 25, 30, 33, 36, 39, 42-dodecaazatetratetracontanedioate (56) .
  • Example 60 Synthesis of (14S, 22S, 23S, 31S) -22, 23-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -4, 7, 10, 13, 16, 21, 24, 29, 32, 35, 38, 41-dodecaoxo-14, 31-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 6, 9, 12, 15, 20, 25, 30, 33, 36, 39, 42-dodecaazatetratetracontanedioic acid (57) .
  • Example 62 Synthesis of tert-butyl ( (benzyloxy) carbonyl) -L-alanyl-L-alaninate (59) .
  • Example 65 Synthesis of tert-butyl ( (benzyloxy) carbonyl) -L-alanyl-L-alanyl-L-alanyl-L-alaninate (62) .
  • Example 66 Synthesis of tert-butyl L-alanyl-L-alanyl-L-alanyl-L-alaninate (63) .
  • Example 67 Synthesis of tert-butyl ( (S) -37- ( ( (benzyloxy) carbonyl) amino) -31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azaoctatriacontan-38-oyl) -L-alanyl-L-alanyl-L-alanyl-L-alaninate (64) .
  • Example 68 Synthesis of tert-butyl ( (S) -37-amino-31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azaoctatriacontan-38-oyl) -L-alanyl-L-alanyl-L-alanyl-L-alaninate (65) .
  • Example 69 Synthesis of di-tert-butyl (2S, 5S, 8S, 11S, 14S, 22S, 23S, 31S, 34S, 37S, 40S, 43S) -22, 23-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -2, 5, 8, 11, 34, 37, 40, 43-octamethyl-4, 7, 10, 13, 16, 21, 24, 29, 32, 35, 38, 41-dodecaoxo-14, 31-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 6, 9, 12, 15, 20, 25, 30, 33, 36, 39, 42-dodecaazatetratetracontanedioate (66) .
  • Example 70 Synthesis of (2S, 5S, 8S, 11S, 14S, 22S, 23S, 31S, 34S, 37S, 40S, 43S) -22, 23-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -2, 5, 8, 11, 34, 37, 40, 43-octamethyl-4, 7, 10, 13, 16, 21, 24, 29, 32, 35, 38, 41-dodecaoxo-14, 31-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 6, 9, 12, 15, 20, 25, 30, 33, 36, 39, 42-dodecaazatetratetracontanedioic acid (67) .
  • MS-ESI (m/z) [M + H] + calcd for C 142 H 203 F 2 N 22 O 50 , 3054.40; found, 3054.90; or68c as a light yellow solid (205.8 mg, 71%yield) .
  • MS-ESI (m/z) [M + H] + calcd for C 150 H 217 F 2 N 24 O 50 , 3192.51; found, 3192.95; .
  • Example 72 Synthesis of di-tert-butyl 5, 5'- ( ( ( (10S, 18S, 19S, 27S) -18, 19-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -4, 9, 12, 17, 20, 25, 28, 33-octaoxo-10, 27-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 8, 11, 16, 21, 26, 29, 34-octaazahexatriacontanedioyl) bis (azanediyl) ) bis (4-hydroxy-3, 1-phenylene) ) (4R, 4'R) -bis (4- ( (tert-butoxycarbonyl) amino) pentanoate) (70) .
  • Example 74 Synthesis of (4R, 4'R) -5, 5'- ( ( ( (10S, 18S, 19S, 27S) -18, 19-bis (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -4, 9, 12, 17, 20, 25, 28, 33-octaoxo-10, 27-bis (31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azahexatriacontan-36-yl) -3, 8, 11, 16, 21, 26, 29, 34-octaazahexatriacontanedioyl) bis (azanediyl) ) bis (4-hydroxy-3, 1-phenylene) ) bis (4- (2- ( (6S, 9R, 11R) -6- ( (S) -sec-butyl) -9-isopropyl-2, 3, 3, 8-tetramethyl-4, 7, 13-trioxo-12-oxa-2, 5, 8-tria
  • Example 76 Synthesis of tert-butyl ( (S) -37- ( ( (benzyloxy) carbonyl) amino) -31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azaoctatriacontan-38-oyl) -L-valyl-L-alaninate (74) .
  • Example 77 Synthesis of ( (S) -37- ( ( (benzyloxy) carbonyl) amino) -31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azaoctatriacontan-38-oyl) -L-valyl-L-alanine (75) .
  • Example 78 Synthesis of 2, 5-dioxopyrrolidin-1-yl ( (S) -37- ( ( (benzyloxy) carbonyl) amino) -31-oxo-2, 5, 8, 11, 14, 17, 20, 23, 26, 29-decaoxa-32-azaoctatriacontan-38-oyl) -L-valyl-L-alaninate (76) .

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Abstract

L'invention concerne un anticorps et un conjugué anticorps-médicament (ADC) comprenant un anticorps monoclonal, ou un fragment de liaison à l'antigène de celui-ci, conjugué à une cytotoxine, dirigé contre l'antigène de maturation des lymphocytes B (BCMA). L'anticorps anti-BCMA et son ADCV sont utiles pour le traitement de cellules de myélome multiple, d'une maladie médiée par les cellules B ou Médiée par les cellules plasmatiques, et de troubles immunitaires ainsi que pour la détection du BCMA. L'invention concerne en outre des compositions pharmaceutiques et des méthodes de traitement médical.
PCT/CN2022/123901 2021-11-03 2022-10-08 Anticorps monoclonal anti-bcma et conjugué anticorps-médicament WO2023078021A1 (fr)

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AU2022383265A AU2022383265A1 (en) 2021-11-03 2022-10-08 Bcma monoclonal antibody and the antibody-drug conjugate
CA3236852A CA3236852A1 (fr) 2021-11-03 2022-10-08 Anticorps monoclonal anti-bcma et conjugue anticorps-medicament
PCT/CN2022/129122 WO2023078273A1 (fr) 2021-11-03 2022-11-02 Conjugaison spécifique pour un conjugué anticorps-médicament
CA3236754A CA3236754A1 (fr) 2021-11-03 2022-11-02 Conjugaison specifique pour un conjugue anticorps-medicament
AU2022381163A AU2022381163A1 (en) 2021-11-03 2022-11-02 Specific conjugation for an antibody-drug conjugate
CN202280069631.1A CN118215676A (zh) 2021-11-03 2022-11-02 抗体药物偶联物的特异性偶联

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AU2022381163A1 (en) 2024-06-13
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AU2022383265A1 (en) 2024-06-13
CA3236852A1 (fr) 2023-05-11
WO2022078524A2 (fr) 2022-04-21
AU2021362997A1 (en) 2024-05-16
CN117980327A (zh) 2024-05-03
CA3236754A1 (fr) 2023-05-11
WO2022078524A3 (fr) 2022-08-25
CN118215676A (zh) 2024-06-18
CA3236930A1 (fr) 2022-04-21

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